# Patent

## Title
Administration of FGF2 for treamtent of anxiety

## Abstract

The present invention provides methods for diagnosing mental disorders. The invention also provides methods of identifying modulators of mental disorders as well as methods of using these modulators to treat patients suffering from mental disorders.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
The present application is related to U.S. Ser. No. 60/581,998, filed Jun. 21, 2004, and U.S. Ser. No. 60/621,252, filed Oct. 22, 2004, and U.S. Ser. No. 60/667,296, filed Mar. 31, 2005 each of which is incorporated herein in its entirety by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Clinical depression, including both bipolar disorders and major depression disorders, is a major public health problem, affecting an estimated 9.5% of the adult population of the United States each year. While it has been hypothesized that mental illness, including mood disorders such as major depression (“MDD”) and bipolar disorder (“BP”) as well as psychotic disorders such as schizophrenia, may have genetic roots, little progress has been made in identifying gene sequences and gene products that play a role in causing these disorders, as is true for many diseases with a complex genetic origin (see, e.g., Burmeister, Biol. Psychiatry 45:522-532 (1999)).
The current lack of biomarkers and the ineffectiveness and reliability of the diagnosis and rates are important issues for the treatment of mental disorders. For example, around 15% of the population suffers from MDD while approximately 1% suffers from BP disorders. Diagnosing bipolar disorder is difficult when, as sometimes occurs, the patient presents only symptoms of depression to the clinician. At least 10-15% of BP patients are reported to be misdiagnosed as MDD. The consequences of such misdiagnosis include a delay in being introduced to efficacious treatment with mood stabilizers and a delay in seeking or obtaining counseling specific to bipolar disorder. Also treatment with antidepressants alone induces rapid cycling, switching to manic or mixed state, and consequently increases suicide risk. Furthermore, in addition to a lack of efficacy, long onset of action and side effects (sexual, sleep, weight gain, etc.), there are recent concerns relating to the undesirable effects of ADs on metabolic syndromes, such as diabetes and hypercholesteremia.
BRIEF SUMMARY OF THE INVENTION
Relying on the discovery that certain genes expressed in particular brain pathways and regions are likely involved in the development of mental illness, the present invention provides methods for diagnosis and treatment of mental illness, as well as methods for identifying compounds effective in treating mental illness.
In order to further understand the neurobiology of mood disorders such as bipolar disorders (BP) and major depression disorders (MDD), the inventors of the present application have used DNA microarrays to study expression profiles of human post-mortem brains from patients diagnosed with BP or MDD. In one aspect, the present invention relates to differential gene expression in the Anterior Cingulate (AnCg), Dorsolateral Prefrontal (DLPFC), and Cerebellar (CB) cortices, Hippocampus, Nucleus Accumbens and Amygdala regions of the brain, wherein the differential gene expression is associated with Bipolar Disorder (BPD) and Major Depressive Disorder (MDD). Certain genes in these regions are considered “unique” to a given disorder such as BP or MDD when differentially expressed in a particular mood disorder and not another (see, e.g., Tables 3, 4, 14-20). In other cases, where the genes are differentially expressed in both BP and MDD relative to healthy controls, the genes are considered to be involved in both disorders. Expression of the differentially expressed genes may be detected using any suitable methods of detection, e.g., microarrays, PCR or in situ hybridization. Gene expression may be detected in brain tissue, brain tissue samples, or other tissue samples (e.g., blood samples in the case of NCAM1).
In one aspect, the present invention relates to differential gene expression associated with G protein-coupled receptors (GPCR)s and downstream signaling pathways, mediated by cyclic adenosine monophosphate (cAMP) and phosphatidylinositol (PI), wherein the gene expression differentially occurs in the Anterior Cingulate (AnCg), Dorsolateral Prefrontal (DLPFC), and Cerebellar (CB) cortices, Hippocampus, Nucleus Accumbens and Amygdala regions of the brains of patients with Bipolar Disorder (BPD) and/or Major Depressive Disorder (MDD), relative to healthy controls (see, e.g., Tables 14-20).
In another aspect, the present invention relates to differential gene expression associated with G protein-coupled receptors (GPCR)s and downstream signaling pathways, mediated by cyclic adenosine monophosphate (cAMP) and phosphatidylinositol (PI), wherein the gene expression differentially occurs in the Hippocampus, Nucleus Accumbens and Amygdala regions of the brains of patients with Bipolar Disorder (BPD) and/or Major Depressive Disorder (MDD), relative to healthy controls (see, e.g., Tables 18-20).
The present invention also demonstrates differential expression of the FGF pathway in the frontal cortex of MDD subjects. Particular FGF-related genes, such as FGF2, are dysregulated by antidepressant therapy, environmental complexity, and the correlation to anxiety-like behavior (see, e.g., Tables 1a, 1b, and 2, and FIGS. 1-7 and 22). The FGF pathway is also related to neurogenesis, e.g., neural stem cell proliferation and differentiation, and the genes disclosed herein can be used for diagnosis and therapeutics related to neurogenesis. Furthermore, FIG. 23 shows the effects of postnatal FGF-2 adminstration on neurogenesis, emotionality and gene expression in adult rats. The FGF injected animals exhibit significantly increased cell survival and proliteration in the dentate gyrus of the hippocampus. As adults, the animals show higher locomotor activity in a novel environment, an index of lower anxiety, and have better learning and memory.
The present invention also demonstrates that the genes of the glutamate/GABA signaling pathways are involved in MDD and BP (see FIG. 24 and Table 8).
The present invention also demonstrates that mitrochondrial genes are involved in MDD andBP (see Table 10).
The present invention also demonstrates that 40 genes encoding growth factor family members and growth factor receptors are significantly differentially expressed in BP or MDD in the DLPFC or AnCg (see Tables 5, 6 and 7).
The present invention demonstrates that genes involved in G protein coupled receptors and their downstream signaling pathways, including cyclic AMP, phosphatidylinositol, and mitogen-activated protein kinase signaling pathways are dysregulated in BP and/or MDD (see Tables 6 and 9 and FIGS. 8-13 and 17-19).
Finally the present invention provides for the first time a novel insertion/deletion polymporphism in the phosphoserine phosphatase-like gene (PSPHL) and demonstrates that a novel deletion polymorphism of PSPHL is related to susceptibility to bipolar disorder. Therefore, detection of this polymorphism is useful for diagnosis of BP, as well as for drug discovery assays for BP therapeutics. In addition, the serine amino acid metabolic pathway, of which PSPHL is a member, is a target for drug discovery for BP therapeutics. The PSPHL gene was first cloned by Planitzer et al., Gene 210 297-306 (1998). The accession number for a representative nucleic acid sequence is AJ0016112 and the accession number for a representative protein sequence is CAA04865.1. See FIGS. 14-16.
The present invention demonstrates, for the first time, unique expression of the 24 nucleic acids listed in Table 3 in the brains of bipolar disorder subjects but not major depression subjects; the unique expression of the 24 nucleic acids listed in Table 4 in the brains of major depression subjects but not bipolar subjects, and the differential and/or unique expression of the nucleic acids listed in Tables 5-10 in the brains of patients suffering from bipolar disorder and major depression disorder, in comparison with normal control subjects. In addition, the present invention identifies biochemical pathways involved in uniquely or differentially in mood disorders, where the proteins encoded by the nucleic acids listed in Table 3-10 are components of the biochemical pathways (e.g., the growth factor, e.g., FGF, signal transduction pathway, GPCR signal transduction pathways, mitochondrial pathways, and glutamate/GABA signaling pathways). Furthermore, the invention demonstrates the unique expression of a PSPHL deletion polymorphism and it's associate with BP.
Genes and pathways that are uniquely or differentially expressed in MDD or BP are useful in diagnosing mood disorders and in assaying for therapeutics that can specifically treat MDD or BP, or can be used to treat both MDD and BP. Differential expression by brain region similarly is a useful diagnostic and therapeutic tool, as certain mood disorders primarily affect certain brain regions. Each brain region plays a unique and critical role in the overall phenotype of any particular mood disorder. Furthermore, because of the relationship between BP and psychotic disorders such as schizo affective disorders, the gene described herein unique to BP can also be uniquely expressed in schizophrenia, and so can be used for differential diagnosis with MDD.
This invention thus provides methods for determining whether a subject has or is predisposed for a mental disorder such as bipolar disorder or major depression disorder. The invention also provides methods of providing a prognosis and for monitoring disease progression and treatment. Furthermore, the present invention provides nucleic acid and protein targets for assays for drugs for the treatment of mental disorders such as bipolar disorder and major depression disorder.
In some embodiments, the methods comprise the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence listed in Tables 3-10 and FIG. 14; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mental disorder.
In some embodiments, the reagent is an antibody. In some embodiments, the reagent is a nucleic acid. In some embodiments, the reagent associates with a polynucleotide. In some embodiments, the reagent associates with a polypeptide. In some embodiments, the polynucleotide comprises a nucleotide sequence listed in Table 3-6. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 3-6. In some embodiments, the level of reagent that associates with the sample is different (i.e., higher or lower) from a level associated with humans without a mental disorder. In some embodiments, the biological sample is obtained from amniotic fluid. In some embodiments, the mental disorder is a mood disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
The invention also provides methods of identifying a compound for treatment of a mental disorder. In some embodiments, the methods comprises the steps of: (i) contacting the compound with a polypeptide, which is encoded by a polynucleotide that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of Table 2, 3, or 4; and (ii) determining the functional effect of the compound upon the polypeptide, thereby identifying a compound for treatment of a mental disorder.
In some embodiments, the contacting step is performed in vitro. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 3-6. In some embodiments, the polypeptide is expressed in a cell or biological sample, and the cell or biological sample is contacted with the compound. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the methods further comprise administering the compound to an animal and determining the effect on the animal, e.g., an invertebrate, a vertebrate, or a mammal. In some embodiments, the determining step comprises testing the animal's mental function.
In some embodiments, the methods comprise the steps of (i) contacting the compound to a cell, the cell comprising a polynucleotide that hybridizes under stringent conditions to a nucleotide sequence of Table 3-6; and (ii) selecting a compound that modulates expression of the polynucleotide, thereby identifying a compound for treatment of a mental disorder. In some embodiments, the polynucleotide comprises a nucleotide sequence listed in Table 3-6. In some embodiment, the expression of the polynucleotide is enhanced. In some embodiments, the expression of the polynucleotide is decreased. In some embodiments, the methods further comprise administering the compound to an animal and determining the effect on the animal. In some embodiments, the determining step comprises testing the animal's mental function. In some embodiments, the mental disorder is a mood disorder or a psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression. In some embodiments, the psychotic disorder is schizophrenia.
The invention also provides methods of treating a mental disorder in a subject. In some embodiments, the methods comprise the step of administering to the subject a therapeutically effective amount of a compound identified using the methods described above. In some embodiments, the mental disorder is a mood disorder or a psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the compound is a small organic molecule, an antibody, an antisense molecule, or a peptide.
The invention also provides methods of treating mental illness in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, which is encoded by a polypeptide that hybridizes under stringent conditions to a nucleic acid of Table 3-6. In some embodiments, the polypeptide comprises an amino acid sequence encoded by a gene listed in Table 3-6. In some embodiments, the mental illness is a mood disorder or a psychotic disorder. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the mood disorder is a bipolar disorder or major depression.
The invention also provides methods of treating mental illness in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, wherein the polypeptide hybridizes under stringent conditions to a nucleic acid of Table 3-6. In some embodiments, the mental illness is a mood disorder or a psychotic disorder. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the mood disorder is a bipolar disorder or major depression.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Rodent FGFR2 ISH. Graph shows mean grayscale (n=6 per group) intensity and standard error bars for 35 S signal for regions indicated for both fluoxetine treated and saline treated rats. The increase in signal in the fluoxetine treated group is significant as determined boy a tow-way ANOVA (treatment, brain region) with p=0.0049, Fisher's PLSD.
FIG. 2: FIG. 2 summarizes differential expression of FGF system transcripts in MDD cortex.
FIG. 3: FIG. 3 shows FGF dysregulation is attenuated by anti-depressant therapy.
FIG. 4: FIG. 4 shows that chronic fluoxetine treatment increases FGFR2 expression in rat forebrain.
FIG. 5: FIG. 5 shows environmental complexity: FGF2 and anxiety-like behavior.
FIG. 6: FIG. 6 shows FGF expression negatively correlates with anxiety-like behavior.
FIG. 7: FIG. 7 shows summarizes FGF and negative affect.
FIG. 8: FIG. 8 shows qRT-PCR validation of microarray data.
FIG. 9: FIG. 9 shows G-protein coupled receptor (GPCR) and ligands dysregulated in anterior cingulated cortex of BP subjects.
FIG. 10: shows gene category over-representation analysis of the three downstream GPCR signaling pathways.
FIG. 11: FIG. 11 shows phosphatidylinositol metabolism in BP disorder.
FIG. 12: FIG. 12 shows mitogen activated protein kinase signaling in BP disorder.
FIG. 13: FIG. 13 shows cAMP signaling pathway in BP subjects
FIG. 14: FIG. 14 shows genomic structure of the PSPHL gene and the deletion polymorphism of PSPHL that is related to BP susceptibility.
FIG. 15: FIG. 15 shows the predicted amino acid sequences for PSPH, PSPHL-A, and PSPHL-B.
FIG. 16: FIG. 16 shows the serine amino acid metabolic pathway.
FIG. 17: FIG. 17 shows where each exon begins and ends in the PSPHL mRNA (SEQ ID NOS:14-20) and provides primers to detect insertion/deletion polymorphisms in the PSPHL locus (SEQ ID NOS:21-24).
FIG. 18: FIG. 18 shows a gel image for PSPHL insertion/deletion alleles.
FIG. 19: FIG. 19 shows the cAMP signaling pathway in the limbic system for BP.
FIG. 20: FIG. 20 shows the PI signaling pathway in the limbic system for MDD.
FIG. 21: FIG. 21 shows MAPK signaling pathway in the limbic system for MDD.
FIG. 22: FIG. 22 shows the effects of environmental complexity on differences in anxiety behavior and FGF2 gene expression.
FIG. 23: FIG. 23 shows the effects of postnatal FGF2 administration on neurogenesis, emotionality and gene expression in adult rats.
FIG. 24: FIG. 24 shows GABA/glutamate signaling pathways in BP and MDD.
FIG. 25: FIG. 25 shows NCAM SNPs and splice variants involved in mood disorders such as bipolar disorder.
FIG. 26: The figure summarizes differential expressed genes regulating cAMP-(A, B) and phosphatidylinositol-(C, D) signaling pathways in the brain of BPD (A, C) and MDD (B, D). GNAI1: G protein alpha inhibiting activity 1, RGS20: Regulator of G-protein signaling 20, PDE1A: Phosphodiesterase 1A, PDE8A: Phosphodiesterase 8A, PKIA: Protein kinase A inhibitor alpha, CDK5: Cyclin-dependent kinase 5, PPP1CA: Protein phosphatase 1, catalytic alpha, PPPlR3C: Protein phosphatase 1, regulatory 3C, INPP5A: Inositol polyphosphate-5-phosphatase A, INPP5F: Inositol polyphosphate-5-phosphatase F, ITPKB: Inositol 1,4,5-trisphosphate 3-kinase B, INPP1: Inositol polyphosphate-1-phosphatase, CDS 1: CDP-diacylglycerol synthase 1, PIK3C2A: Phosphoinositide-3-kinase catalytic 2A, PIK3C2B: Phosphoinositide-3-kinase catalytic 2B, PIK3R1: Phosphoinositide-3-kinase regulatory 1, PRKCI: Protein kinase C iota, ITPR1: Inositol 1,4,5-triphosphate receptor 1, PRKB 1: Protein kinase C beta 1, NPY: Neuropeptide Y, SST: Somatostatin, NPY1R: Neuropeptide Y receptor Y1, TACR2: Tachykinin receptor 2, NTSR2: Neurotensin receptor 2, EDNRB: Endothelin receptor type B, GRM3: Metabotropic Glutamate receptor 3, EDG1: Endothelial differentiation GPCR 1, EDG2: Endothelial differentiation GPCR 2.
FIG. 27: In situ hybridization images of GPR37 mRNA in representative BPD, MDD and control subjects GPR37 mRNA is preferentially expressed in subcortical white matter. Among 6 layers of cortical gray matter, GPR37 expression in deeper layer (V-VI) is relatively higher than superficial layer (I-III). Expression levels in GPR37 is increased in the BPD subjects, and decreased in MDD subjects in subcortical white matter in anterior cingulate cortex tissue, compared to control subjects. WM: Subcortical white matter, BPD: Bipolar disorder, MDD: Major depressive disorder.
FIGS. 28 and 29: In situ hybridization for LRPPRC (leucine-rich PPR-motif containing) mRNA in three brain regions. (A). LRPPRC expression in BPD and control representative images. (B). Controls with agonal factors showed a 36% reduction in LRPPRC compared to controls with zero agonal factors (p=0.011) in cerebellum and a similar effect was seen across the cortical regions. (C). In DLPFC, the BPD cases without agonal factors show increased LRPPRC compared to controls without agonal factors (p=0.001) and compared to MDD subjects (p=0.02) without agonal factors.
FIG. 30: NCAM1 (i.e., neural cell adhesion molecule 1) genomic organization and location of four polymorphic sites. The gene spans 214 kb, but does not contain any exonic SNPs. The arrows indicate the location of the four polymorphisms and the five exons used in this exploratory analysis.
FIG. 31: Significant alterations of NCAMI exon splice variant levels are shown by genotype and diagnosis by genotype.



TABLE LEGENDS
Table 1a: Table 1a lists subject data for cohort A.
Table 1b: Table 1b lists subject data for cohort B.
Table 2: Table 2 shows microarray data for all FGF transcripts detected in either DLPFC or AnCg and summary data for confirmation studies.
Table 3: Table 3 lists genes uniquely expressed in BP subjects.
Table 4: Table 4 lists genes uniquely expressed in MDD subjects.
Table 5: Table 5 lists growth factor pathway genes expressed in MDD and BP subjects.
Table 6: Table 6 lists GPCR pathway genes expressed in MDD and BP subjects.
Table 7: Table 7 lists growth factor pathway genes expressed in MDD and BP subjects.
Table 8: Table 8 lists GABA and glutamate signaling pathway genes expressed in MDD and BP subjects.
Table 9: Table 9 lists GPCR pathway genes expressed in MDD and BP subjects.
Table 10: Table 10 lists mitochondrial genes expressed in MDD and BP subjects.
Table 11: Table 11 lists genes expressed in MDD, BP, and schizophrenia subjects.
Table 12: Table 12 lists genes expressed in MDD, BP, and schizophrenia subjects.
Table 13: Table 13 lists GPCR pathway genes expressed in MDD and BP.
Table 14: GPCRs and related signaling genes dysregulated in anterior cingulate cortex.
Table 15: GPCRs and related signaling genes dysregulated in dorsolateral prefrontal cortex.
Table 16: GPCRs and related signaling genes dysregulated in cerebellar cortex.
Table 17: Quantitative RT-PCR data. Fold changes in microarray and qRT-PCR analyses for representative ligand peptides, GPCRs, G protein regulator (NPY, SST, GPR37, GPRC5B, RGS20), which were dysregulated in BPD/MDD compared to the control group. N.S., No significant change; *. p<0.05; **, p<0.01.
Table 18: GPCRs and related signaling genes dysregulated in amygdala, hippocampus, nucleus accumbens of BPD.
Table 19: GPCRs and related signaling genes dysregulated in amygdala, hippocampus, nucleus accumbens of MDD.
Table 20: Table 20 shows the genes that were differentially expressed in BPD or MDD by >1.2 fold change and were down-regulated in agonal factor control comparisons by <1.0. The opposite genes are also shown, where there was a decrease in mood disorder by <−1.2 fold change, and the agonal factor control comparison showed an increase >1.0 fold change. These genes were found in 4 major classifications listed: mitochondria, chaperone, apoptosis, and proteasome.
Table 21: Real time Q-PCR validation results for selected mitochondrial related candidate genes for mood disorders in two cortical regions. These genes are nuclear-encoded. Significant by Q-PCR p<0.05 one-tailed t-test. The Q-PCR t-test MDD, BPD, and control groups used subjects with no agonal factors and pH>6.8 similar to microarray analysis #3 groups MDD-High, BPD-High, and Control-High.
Table 22: Mitochondrial DNA (mtDNA) encoded genes were analyzed by real time Q-PCR for differential expression in BPD and MDD compared to controls. Nuclear encoded genes in BPD and MDD subjects appeared to generally be increased while several mtDNA genes showed a significant decrease by Q-PCR in mood disorders.
Table 23: Primers for each DNA segment and possible combination of splice variants (a, b, c, SEC and VASE) (SEQ ID NOS:1-11), as well as, for SNP 9 (SEQ ID NOS:12-13) and an exon outside of the splice sites for measuring total NCAM1. The numbering is shown in FIG. 26 according to accession M22094. *1—Only the forward primer could be designed because exon a is 14 bps. 2—Exon 3 is before the variable exons and only the forward primer was needed to PCR outside the exons. 3—Exon 8 is after the variable exons and only the reverse primer was needed to PCR outside the exons.
Table 24: Genotypic Association Results. The odds ratio, chi-square (chi2) and p-values where all calculated using the DeFinetti program Tests for Deviation from HWE and Tests for Association (C.I.: 95% confidence interval).
Table 25: SNP 9 and SNP b haplotype frequency, odds ratio and p-values. P-values were calculated from the Chi-squared values derived from the EHplus program.
Table 26: Genotypic and Allelic Distributions for Controls, Bipolar Disorder and Schizophrenia. Fisher's exact p-values are shown for allelic distribution between case-controls.
Table 27: Genotype×Splice Variant Differences×Diagnosis (p-values). For each SNP genotype and splice variant the splice variant amounts were evaluated by t-test based on diagnosis and the significant p-values were reported.
Tables 28: Genes upregulated (28.1) and downregulated (28.2) by Lithium in monkey brains.
Table 29: Values of V-ATPase Subunits differential expression in Non-human primate model of depression.
Table 30: Genes differentially expressed in the frontal cortex of rats subjected to chronic unpredictable stress (CUS) and antidepressant administration (All=fluoxetine, desipramine, and bupropion). Controls were administered water (H2O treated).
DEFINITIONS
A “mental disorder” or “mental illness” or “mental disease” or “psychiatric or neuropsychiatric disease or illness or disorder” refers to mood disorders (e.g., major depression, mania, and bipolar disorders), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, anxiety disorders (e.g., obsessive-compulsive disorder) as well as other mental disorders such as substance-related disorders, childhood disorders, dementia, autistic disorder, adjustment disorder, delirium, multi-infarct dementia, and Tourette's disorder as described in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV). Typically, such disorders have a complex genetic and/or a biochemical component.
A “mood disorder” refers to disruption of feeling tone or emotional state experienced by an individual for an extensive period of time. Mood disorders include major depression disorder (i.e., unipolar disorder), mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and many others. See, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
“Major depression disorder,” “major depressive disorder,” or “unipolar disorder” refers to a mood disorder involving any of the following symptoms: persistent sad, anxious, or “empty” mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being “slowed down”; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability; or persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain. Various subtypes of depression are described in, e.g., DSM IV.
“Bipolar disorder” is a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing from being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression), see, e.g., DSM IV.
“A psychotic disorder” refers to a condition that affects the mind, resulting in at least some loss of contact with reality. Symptoms of a psychotic disorder include, e.g., hallucinations, changed behavior that is not based on reality, delusions and the like. See, e.g., DSM IV. Schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, substance-induced psychotic disorder, and shared psychotic disorder are examples of psychotic disorders.
“Schizophrenia” refers to a psychotic disorder involving a withdrawal from reality by an individual. Symptoms comprise for at least a part of a month two or more of the following symptoms: delusions (only one symptom is required if a delusion is bizarre, such as being abducted in a space ship from the sun); hallucinations (only one symptom is required if hallucinations are of at least two voices talking to one another or of a voice that keeps up a running commentary on the patient's thoughts or actions); disorganized speech (e.g., frequent derailment or incoherence); grossly disorganized or catatonic behavior; or negative symptoms, i.e., affective flattening, alogia, or avolition. Schizophrenia encompasses disorders such as, e.g., schizoaffective disorders. Diagnosis of schizophrenia is described in, e.g., DSM IV. Types of schizophrenia include, e.g., paranoid, disorganized, catatonic, undifferentiated, and residual.
An “antidepressant” refers to an agents typically used to treat clinical depression. Antidepressants includes compounds of different classes including, for example, specific serotonin reuptake inhibitors (e.g., fluoxetine), tricyclic antidepressants (e.g., desipramine), and dopamine reuptake inhibitors (e.g, bupropion). Typically, antidepressants of different classes exert their therapeutic effects via different biochemical pathways. Often these biochemical pathways overlap or intersect. Additonal diseases or disorders often treated with antidepressants include, chronic pain, anxiety disorders, and hot flashes.
An “agonist” refers to an agent that binds to a polypeptide or polynucleotide of the invention, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide or polynucleotide of the invention.
An “antagonist” refers to an agent that inhibits expression of a polypeptide or polynucleotide of the invention or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of a polypeptide or polynucleotide of the invention.
“Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term “modulator” includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity. Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
The term “test compound” or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi, oligonucleotide, etc. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
A “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.
An “siRNA” or “RNAi” refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. “siRNA” or “RNAi” thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
“Determining the functional effect” refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a polynucleotide or polypeptide of the invention (such as a polynucleotide of Table 3-6 or a polypeptide encoded by a gene of Table 3-6), e.g., measuring physical and chemical or phenotypic effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand binding affinity; measurement of calcium influx; measurement of the accumulation of an enzymatic product of a polypeptide of the invention or depletion of an substrate; measurement of changes in protein levels of a polypeptide of the invention; measurement of RNA stability; G-protein binding; GPCR phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca2+); identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
Samples or assays comprising a nucleic acid or protein disclosed herein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
“Biological sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, sputum, tissue, lysed cells, brain biopsy, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
“Antibody” refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
The terms “peptidomimetic” and “mimetic” refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the polynucleotides, polypeptides, antagonists or agonists of the invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” (Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as a CCX CKR, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of, e.g., —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO—. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. For example, a mimetic composition is within the scope of the invention if it is capable of carrying out the binding or enzymatic activities of a polypeptide or polynucleotide of the invention or inhibiting or increasing the enzymatic activity or expression of a polypeptide or polynucleotide of the invention.
The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
The term “isolated,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another:

    1) Alanine (A), Glycine (G);
    2) Aspartic acid (D), Glutamic acid (E);
    3) Asparagine (N), Glutamine (Q);
    4) Arginine (R), Lysine (K);
    5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
    6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
    7) Serine (S), Threonine (T); and
    8) Cysteine (C), Methionine (M)
    (see, e.g., Creighton, Proteins (1984)).


“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. Nucleic acids that hybridize to the genes listed in Tables 3-10 and FIG. 14 are encompassed by the invention.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990).
The phrase “a nucleic acid sequence encoding” refers to a nucleic acid that contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
The phrase “specifically (or selectively) binds to an antibody” or “specifically (or selectively) immunoreactive with”, when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.
One who is “predisposed for a mental disorder” as used herein means a person who has an inclination or a higher likelihood of developing a mental disorder when compared to an average person in the general population.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
To understand the genetic basis of mental disorders, studies have been conducted to investigate the expression patterns of genes that are differentially expressed specifically in central nervous system of subjects with mood disorders. Differential and unique expression of known and novel genes was determined by way of interrogating total RNA samples purified from postmortem brains of BP and MDD patients with Affymetrix Gene Chips® (containing high-density oligonucleotide probe set arrays). The fundamental principles is that by identifying genes and pathways that are differentially expressed in BP and/or MDD (relative to healthy control subjects), via global expression profiling of the transcriptomes as above, one can identify genes that cause, effect, or are associated with the disease, or that interact with drugs used to treat the disease, for use in diagnostic and therapeutic applications.
The present invention therefore demonstrates the altered expression (either higher or lower expression as indicated herein) and in some cases unique differential expression of the genes of Tables 3-10 at the mRNA level in selected brain regions of patients diagnosed with mood disorders, as well as the PSPHL gene (see, e.g., FIG. 14) (e.g., bipolar disorder and major depression disorder) in comparison with normal individuals. This invention thus provides methods for diagnosis of mental disorders such as mood disorders (e.g., bipolar disorder, major depression, and the like) and other mental disorders having a genetic component by detecting the level of a transcript or translation product of the genes listed in Tables 3-10 and FIG. 14 as well as their corresponding biochemical pathways.
In one embodiment, the present invention relates to a novel insertion-deletion polymorphism of phosphoserine phosphatase-like gene, and the association between deletion allele of PSPHL and susceptibility to bipolar disorder (BPD). The fact that PSPHL shows dichotomous present/absent pattern of expression among individuals with brain-wide consistency suggests genetic variation in its regulation (see Example 2). Most intriguingly, we have identified an insertion/deletion polymorphism at the PSPHL locus. The deleted genomic region spans more than 30 kb, including the promoter region and the exons 1, 2 and 3 of PSPHL gene. This genetic variance explains the present/absent pattern of the PSPHL expression. An over-representation of the deletion allele resulting in the absence of PSPHL expression increases susceptibility to BPD. The invention therefore provides the first evidence linking a genetic variant of the PSPHL gene to bipolar disorder. The finding will facilitate characterization of the physiological and pathological function of the gene relevant to bipolar disorder, and provides novel and significant use of this gene and its variants for diagnosis, treatment and prevention of bipolar disorder.
The invention further provides methods of identifying a compound useful for the treatment of such disorders by selecting compounds that modulates the functional effect of the translation products or the expression of the transcripts described herein. The invention also provides for methods of treating patients with such mental disorders, e.g., by administering the compounds of the invention or by gene therapy.
The genes and the polypeptides that they encode, which are associated with mood disorders such as bipolar disease and major depression, are useful for facilitating the design and development of various molecular diagnostic tools such as GeneChipsTM containing probe sets specific for all or selected mental disorders, including but not limited to mood disorders, and as an ante-and/or post-natal diagnostic tool for screening newborns in concert with genetic counseling. Other diagnostic applications include evaluation of disease susceptibility, prognosis, and monitoring of disease or treatment process, as well as providing individualized medicine via predictive drug profiling systems, e.g., by correlating specific genomic motifs with the clinical response of a patient to individual drugs. In addition, the present invention is useful for multiplex SNP and haplotype profiling, including but not limited to the identification of therapeutic, diagnostic, and pharmacogenetic targets at the gene, mRNA, protein, and pathway level. Profiling of splice variants and deletions is also useful for diagnostic and therapeutic applications.
The genes and the polypeptides that they encode, described herein, are also useful as drug targets for the development of therapeutic drugs for the treatment or prevention of mental disorders, including but not limited to mood disorders.
Antidepressants belong to different classes, e.g., desipramine, bupropion, and fluoxetine are in general equally effective for the treatment of clinical depression, but act by different mechanisms. The similar effectiveness of the drugs for treatment of mood disorders suggests that they act through a presently unidentified common pathway. Animal models of depression, including treatment of animals with known therapeutics such as SSRIs, can be used to examine the mode of action of the genes of the invention. Lithium is drug of choice for treating BP.
The genes and the polypeptides that they encode, described herein, as also useful as drug targets for the development of therapeutic drugs for the treatment or prevention of mental disorders, including but not limited to mood disorders. Mental disorders have a high co-morbidity with other neurological disorders, such as Parkinson's disease or Alzheimer's. Therefore, the present invention can be used for diagnosis and treatment of patients with multiple disease states that include a mental disorder such as a mood disorder. These mood disorders include BP, MDD, and other disorders such as psychotic-depression, depression and anxiety features, melancholic depression, chronic depression, BPI and BPII.
II. General Recombinant Nucleic Acid Methods for Use with the Invention
In numerous embodiments of the present invention, polynucleotides of the invention will be isolated and cloned using recombinant methods. Such polynucleotides include, e.g., those listed in Tables 3-10 and FIG. 14, which can be used for, e.g., protein expression or during the generation of variants, derivatives, expression cassettes, to monitor gene expression, for the isolation or detection of sequences of the invention in different species, for diagnostic purposes in a patient, e.g., to detect mutations or to detect expression levels of nucleic acids or polypeptides of the invention. In some embodiments, the sequences of the invention are operably linked to a heterologous promoter. In one embodiment, the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, a primate, etc.
A. General Recombinant Nucleic Acids Methods
This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).
B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding Desired Proteins
In general, the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA. The particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences of the genes listed in Tables 3-10 and FIG. 14, which provide a reference for PCR primers and defines suitable regions for isolating specific probes. Alternatively, where the sequence is cloned into an expression library, the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against a polypeptide comprising an amino acid sequence encoded by a gene listed in Table 1-8.
Methods for making and screening genomic and cDNA libraries are well known to those of skill in the art (see, e.g., Gubler and Hoffman Gene 25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); and Sambrook, supra). Brain cells are an example of suitable cells to isolate RNA and cDNA sequences of the invention.
Briefly, to make the cDNA library, one should choose a source that is rich in mRNA. The mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. For a genomic library, the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template. Suitable primers can be designed from specific sequences of the invention. This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
Appropriate primers and probes for identifying polynucleotides of the invention from mammalian tissues can be derived from the sequences provided herein. For a general overview of PCR, see, Innis et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990).
Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.
A gene encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes, using standard methods well known to those of skill in the art, or eukaryotes as described infra.
III. Purification of Proteins of the Invention
Either naturally occurring or recombinant polypeptides of the invention can be purified for use in functional assays. Naturally occurring polypeptides, e.g., polypeptides encoded by genes listed in Tables 3-10 and FIG. 14, can be purified, for example, from mouse or human tissue such as brain or any other source of an ortholog. Recombinant polypeptides can be purified from any suitable expression system.
The polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
A number of procedures can be employed when recombinant polypeptides are purified. For example, proteins having established molecular adhesion properties can be reversible fused to polypeptides of the invention. With the appropriate ligand, the polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the polypeptide can be purified using immunoaffinity columns.
A. Purification of Proteins from Recombinant Bacteria
When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al., both supra, and will be apparent to those of skill in the art.
The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.
Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques.
Alternatively, it is possible to purify proteins from bacteria periplasm. Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al., supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
B. Standard Protein Separation Techniques for Purifying Proteins
1. Solubility Fractionation
Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
2. Size Differential Filtration
Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
3. Column Chromatography
The proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art.
It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
IV. Detection of Gene Expression
Those of skill in the art will recognize that detection of expression of polynucleotides of the invention has many uses. For example, as discussed herein, detection of the level of polypeptides or polynucleotides of the invention in a patient is useful for diagnosing mood disorders or psychotic disorders or a predisposition for a mood disorder or psychotic disorders. Moreover, detection of gene expression is useful to identify modulators of expression of the polypeptides or polynucleotides of the invention.
A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.
The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969).
Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, “Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20).
The probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind. Thus, the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3H, 125I, 35S, 14C, or 32P-labeled probes or the like.
Other labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).
In general, a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
Most typically, the amount of RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantifying labels are well known to those of skill in the art.
In preferred embodiments, the target nucleic acid or the probe is immobilized on a solid support. Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.
A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer arrays (VLSIPSTM), available from Affymetrix, Inc. (Santa Clara, Calif.) can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759.
Detection can be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes). One preferred example uses an antibody that recognizes DNA-RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Coutlee et al. (1989) Analytical Biochemistry 181:153-162; Bogulavski (1986) et al. J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc. Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).
In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies that are commercially or publicly available. In addition to the art referenced above, general methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art (see, e.g., Paul (3rd ed.) Fundamental Immunology Raven Press, Ltd., NY (1993); Coligan Current Protocols in Immunology Wiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY (1988); Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497 (1975)). Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al. Nature 341:544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 μM, preferably at least about 0.01 μM or better, and most typically and preferably, 0.001 μM or better.
The nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target. For example, the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system, in particular RT-PCR or real time PCR, and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.
V. Immunological Detection of the Polypeptides of the Invention
In addition to the detection of polynucleotide expression using nucleic acid hybridization technology, one can also use immunoassays to detect polypeptides of the invention. Immunoassays can be used to qualitatively or quantitatively analyze polypeptides. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
A. Antibodies to Target Polypeptides or Other Immunogens
Methods for producing polyclonal and monoclonal antibodies that react specifically with a protein of interest or other immunogen are known to those of skill in the art (see, e.g., Coligan, supra; and Harlow and Lane, supra; Stites et al., supra and references cited therein; Goding, supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors (see, Huse et al., supra; and Ward et al., supra). For example, in order to produce antisera for use in an immunoassay, the protein of interest or an antigenic fragment thereof, is isolated as described herein. For example, a recombinant protein is produced in a transformed cell line. An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen.
Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross-reactivity against unrelated proteins or even other homologous proteins from other organisms, using a competitive binding immunoassay. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better.
A number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein, such as one comprising an amino acid sequence encoded by a gene listed in Table 1-8 may also be used either in pure or impure form. Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow and Lane, supra).
Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., supra.
Once target protein specific antibodies are available, the protein can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician. For a review of immunological and immunoassay procedures in general see, Stites, supra. Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra; and Harlow and Lane, supra.
Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to the protein (e.g., one has an amino acid sequence encoded by a gene listed in Table 1-8) or a fragment thereof. This antiserum is selected to have low cross-reactivity against different proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.
B. Immunological Binding Assays
In a preferred embodiment, a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites, supra. Immunological binding assays (or immunoassays) typically utilize a “capture agent” to specifically bind to and often immobilize the analyte (in this case a polypeptide of the present invention or antigenic subsequences thereof). The capture agent is a moiety that specifically binds to the analyte. In a preferred embodiment, the capture agent is an antibody that specifically binds, for example, a polypeptide of the invention. The antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
In a preferred embodiment, the labeling agent is a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval, et al. J. Immunol., 111: 1401-1406 (1973); and Akerstrom, et al. J. Immunol., 135:2589-2542 (1985)).
Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10° C. to 40° C.
1. Non-Competitive Assay Formats
Immunoassays for detecting proteins of interest from tissue samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein) is directly measured. In one preferred “sandwich” assay, for example, the capture agent (e.g., antibodies specific for a polypeptide encoded by a gene listed in Table 1-8) can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample. The polypeptide thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
2. Competitive Assay Formats
In competitive assays, the amount of analyte (such as a polypeptide encoded by a gene listed in Table 1-8) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (e.g., an antibody specific for the analyte) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, the protein of interest is added to the sample and the sample is then contacted with a capture agent, in this case an antibody that specifically binds to a polypeptide of the invention. The amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. For example, the amount of the polypeptide bound to the antibody may be determined either by measuring the amount of subject protein present in a protein/antibody complex or, alternatively, by measuring the amount of remaining uncomplexed protein. The amount of protein may be detected by providing a labeled protein molecule.
Immunoassays in the competitive binding format can be used for cross-reactivity determinations. For example, a protein of interest can be immobilized on a solid support. Proteins are added to the assay which compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein of interest. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.
The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein.
3. Other Assay Formats
In a particularly preferred embodiment, western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest. For example, the antibodies specifically bind to a polypeptide of interest on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.
Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev. 5:34-41).
4. Labels
The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
Non-radioactive labels are often attached by indirect means. The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound. A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Pat. No. 4,391,904).
Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.
In some embodiments, BP or MDD in a patient may be diagnosed or otherwise evaluated by visualizing expression in situ of one or more of the appropriately dysregulated gene sequences identified herein. Those skilled in the art of visualizing the presence or expression of molecules including nucleic acids, polypeptides and other biochemicals in the brains of living patients will appreciate that the gene expression information described herein may be utilized in the context of a variety of visualization methods. Such methods include, but are not limited to, single-photon emission-computed tomography (SPECT) and positron-emitting tomography (PET) methods. See, e.g., Vassaux and Groot-wassink, “In Vivo Noninvasive Imaging for Gene Therapy,” J. Biomedicine and Biotechnology, 2: 92-101 (2003).
PET and SPECT imaging shows the chemical functioning of organs and tissues, while other imaging techniques—such as X-ray, CT and MRI—show structure. The use of PET and SPECT imaging is useful for qualifying and monitoring the development of brain diseases, including schizophrenia and related disorders. In some instances, the use of PET or SPECT imaging allows diseases to be detected years earlier than the onset of symptoms. The use of small molecules for labelling and visualizing the presence or expression of polypeptides and nucleotides has had success, for example, in visualizing proteins in the brains of Alzheimer's patients, as described by, e.g., Herholz K et al., Mol Imaging Biol., 6(4):239-69 (2004); Nordberg A, Lancet Neurol., 3(9):519-27 (2004); Neuropsychol Rev., Zakzanis K K et al., 13(1):1-18 (2003); Kung M P et al, Brain Res.,1025(1-2):98-105 (2004); and Herholz K, Ann Nucl Med., 17(2):79-89 (2003).
The dysregulated genes disclosed in Tables 1-30, or their encoded peptides (if any), or fragments thereof, can be used in the context of PET and SPECT imaging applications. After modification with appropriate tracer residues for PET or SPECT applications, molecules which interact or bind with the transcripts in Tables 1-30 or with any polypeptides encoded by those transcripts may be used to visualize the patterns of gene expression and facilitate diagnosis of schizophrenia MDD or BP, as described herein. Similarly, if the encoded polypeptides encode enzymes, labeled molecules which interact with the products of catalysis by the enzyme may be used for the in vivo imaging and diagnostic application described herein.
Antisense technology is particularly suitable for detecting the the transcripts identified in Tables 1-30 herein. For example, the use of antisense peptide nucleic acid (PNA) labeled with an appropriate radionuclide, such as 111In, and conjugated to a brain drug-targeting system to enable transport across biologic membrane barriers, has been demonstrated to allow imaging of endogenous gene expression in brain cancer. See Suzuki et al., Journal of Nuclear Medicine, 10:1766-1775 (2004). Suzuki et al. utilize a delivery system comprising monoclonal antibodies that target transferring receptors at the blood-brain barrier and facilitate transport of the PNA across that barrier. Modified embodiments of this technique may be used to target upregulated genes associated with schizophrenia, BP or MDD, such as the upregulated genes which appear in Tables 1-30, in methods of treating schizophrenic, BP or MDD patients.
In other embodiments, the dysregulated genes listed in Tables 1-30 may be used in the context of prenatal and neonatal diagnostic methods. For example, fetal or neonatal samples can be obtained and the expression levels of appropriate transcripts (e.g., the transcripts in Table 19) may be measured and correlated with the presence or increased likelihood of a mental disorder, e.g., MDD. Similarly, the presence of one or more of the SNPs identified in the Tables, e.g., Table 27 may be used to infer or corroborate dysregulated expression of a gene and the likelihood of a mood disorder in prenatal, neonatal, children and adult patients.
In other embodiments, the brain labeling and imaging techniques described herein or variants thereof may be used in conjunction with any of the dysregulated gene sequences in Tables 1-30 in a forensic analysis, i.e., to determine whether a deceased individual suffered from schizophrenia, BP, or MDD.
VI. Screening for Modulators of Polypeptides and Polynucleotides of the Invention
Modulators of polypeptides or polynucleotides of the invention, i.e. agonists or antagonists of their activity or modulators of polypeptide or polynucleotide expression, are useful for treating a number of human diseases, including mood disorders or psychotic disorders. Administration of agonists, antagonists or other agents that modulate expression of the polynucleotides or polypeptides of the invention can be used to treat patients with mood disorders or psychotic disorders.
A. Screening Methods
A number of different screening protocols can be utilized to identify agents that modulate the level of expression or activity of polypeptides and polynucleotides of the invention in cells, particularly mammalian cells, and especially human cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that modulates the polypeptide activity by binding to a polypeptide of the invention, modulating inhibitor binding to the polypeptide or activating expression of the polypeptide or polynucleotide, for example.
1. Binding Assays
Preliminary screens can be conducted by screening for agents capable of binding to a polypeptide of the invention, as at least some of the agents so identified are likely modulators of polypeptide activity. The binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet and Yamamura, (1985) “Neurotransmitter, Hormone or Drug Receptor Binding Methods,” in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89. The protein utilized in such assays can be naturally expressed, cloned or synthesized.
Binding assays are also useful, e.g., for identifying endogenous proteins that interact with a polypeptide of the invention. For example, antibodies, receptors or other molecules that bind a polypeptide of the invention can be identified in binding assays.
2. Expression Assays
Certain screening methods involve screening for a compound that up or down-regulates the expression of a polypeptide or polynucleotide of the invention. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polypeptide or polynucleotide of the invention and then detecting an increase or decrease in expression (either transcript, translation product, or catalytic product). Some assays are performed with peripheral cells, or other cells, that express an endogenous polypeptide or polynucleotide of the invention.
Polypeptide or polynucleotide expression can be detected in a number of different ways. As described infra, the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques. Alternatively, a polypeptide of the invention can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to a polypeptide of the invention.
Other cell-based assays are reporter assays conducted with cells that do not express a polypeptide or polynucleotide of the invention. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter of a polynucleotide of the invention that is operably linked to a reporter gene that encodes a detectable product. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, 62 -glucuronidase, chloramphenicol acetyl transferase (CAT); Alton and Vapnek (1979) Nature 282:864-869), luciferase, β-galactosidase, green fluorescent protein (GFP) and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either activates the promoter by binding to it or triggers a cascade that produces a molecule that activates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of a polynucleotide of the invention and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.
The level of expression or activity can be compared to a baseline value. As indicated above, the baseline value can be a value for a control sample or a statistical value that is representative of expression levels for a control population (e.g., healthy individuals not having or at risk for mood disorders or psychotic disorders). Expression levels can also be determined for cells that do not express a polynucleotide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.
A variety of different types of cells can be utilized in the reporter assays. Cells that express an endogenous polypeptide or polynucleotide of the invention include, e.g., brain cells, including cells from the cerebellum, anterior cingulate cortex, dorsolateral prefrontal cortex, amygdala, hippocampus, or nucleus accumbens. Cells that do not endogenously express polynucleotides of the invention can be prokaryotic, but are preferably eukaryotic. The eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs. Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cell lines.
Various controls can be conducted to ensure that an observed activity is authentic including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound. Compounds can also be further validated as described below.
3. Catalytic Activity
Catalytic activity of polypeptides of the invention can be determined by measuring the production of enzymatic products or by measuring the consumption of substrates. Activity refers to either the rate of catalysis or the ability to the polypeptide to bind (Km) the substrate or release the catalytic product (Kd).
Analysis of the activity of polypeptides of the invention are performed according to general biochemical analyses. Such assays include cell-based assays as well as in vitro assays involving purified or partially purified polypeptides or crude cell lysates. The assays generally involve providing a known quantity of substrate and quantifying product as a function of time.
4. Validation
Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Preferably such studies are conducted with suitable animal models. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expression or activity of a polynucleotide or polypeptide of the invention is in fact upregulated. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats. As described herein, models using admininstration of known therapeutics can be useful.
5. Animal Models
Animal models of mental disorders also find use in screening for modulators. In one embodiment, invertebrate models such as Drosophila models can be used, screening for modulators of Drosophila orthologs of the human genes disclosed herein. In another embodiment, transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence, decreased or increased expression of a polynucleotide or polypeptide of the invention. The same technology can also be applied to make knockout cells. When desired, tissue-specific expression or knockout of a polynucleotide or polypeptide of the invention may be necessary. Transgenic animals generated by such methods find use as animal models of mental illness and are useful in screening for modulators of mental illness.
Knockout cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into an endogenous gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting an endogenous polynucleotide of the invention with a mutated version of the polynucleotide, or by mutating an endogenous polynucleotide, e.g., by exposure to carcinogens.
For development of appropriate stem cells, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
B. Modulators of Polypeptides or Polynucleotides of the Invention
The agents tested as modulators of the polypeptides or polynucleotides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Alternatively, modulators can be genetically altered versions of a polypeptide or polynucleotide of the invention. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Modulators also include agents designed to reduce the level of mRNA of the invention (e.g. antisense molecules, ribozymes, DNAzymes and the like) or the level of translation from an mRNA.
In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).
C. Solid State and Soluble High Throughput Assays
In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.
The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-Gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank and Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
The invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of the polynucleotides or polypeptides of the invention. In a preferred embodiment, the methods of the invention include such a control reaction. For each of the assay formats described, “no modulator” control reactions that do not include a modulator provide a background level of binding activity.
In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. First, a known activator of a polynucleotide or polypeptide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of polynucleotide or polypeptide determined according to the methods herein. Second, a known inhibitor of a polynucleotide or polypeptide of the invention can be added, and the resulting decrease in signal for the expression or activity can be similarly detected.
D. Computer-Based Assays
Yet another assay for compounds that modulate the activity of a polypeptide or polynucleotide of the invention involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of the polypeptide or polynucleotide based on the structural information encoded by its amino acid or nucleotide sequence. The input sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the molecule. Similar analyses can be performed on potential receptors or binding partners of the polypeptides or polynucleotides of the invention. The models of the protein or nucleotide structure are then examined to identify regions of the structure that have the ability to bind, e.g., a polypeptide or polynucleotide of the invention. These regions are then used to identify polypeptides that bind to a polypeptide or polynucleotide of the invention.
The three-dimensional structural model of a protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a potential receptor into the computer system. The amino acid sequences encoded by the nucleic acid sequences provided herein represent the primary sequences or subsequences of the proteins, which encode the structural information of the proteins. At least 10 residues of an amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary, and quaternary structure of the protein of interest. The software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as “energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
Once the structure has been generated, potential ligand binding regions are identified by the computer system. Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential ligand is then compared to that of a polypeptide or polynucleotide of the invention to identify binding sites of the polypeptide or polynucleotide of the invention. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of genes encoding a polypeptide or polynucleotide of the invention. Such mutations can be associated with disease states or genetic traits and can be used for diagnosis. As described above, GeneChipTM and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated a polypeptide or polynucleotide of the invention involves receiving input of a first amino acid sequence of a polypeptide of the invention (or of a first nucleic acid sequence encoding a polypeptide of the invention), e.g., any amino acid sequence having at least 60%, optionally at least 70% or 85%, identity with the amino acid sequence of interest, or conservatively modified versions thereof. The sequence is entered into the computer system as described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence. The second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified. Such sequences can represent allelic differences in various polynucleotides of the invention, and mutations associated with disease states and genetic traits.
VII. Compositions, Kits and Integrated Systems
The invention provides compositions, kits and integrated systems for practicing the assays described herein using polypeptides or polynucleotides of the invention, antibodies specific for polypeptides or polynucleotides of the invention, etc.
The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more polynucleotides or polypeptides of the invention immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of polynucleotides or polypeptides of the invention can also be included in the assay compositions.
The invention also provides kits for carrying out the therapeutic and diagnostic assays of the invention. The kits typically include a probe that comprises an antibody that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe. The kits may include several polynucleotide sequences encoding polypeptides of the invention. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding the polypeptides of the invention, or on activity of the polypeptides of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of polypeptides of the invention, a robotic armature for mixing kit components or the like.
The invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of the polypeptides of the invention. The systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.
A number of robotic fluid transfer systems are available, or can easily be made from existing components. For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous STAT binding assays.
Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel ×86 or Pentium chip-compatible DOS®, OS2® WINDOWS®, WINDOWS NT®, WINDOWS95®, WINDOWS98®, or WINDOWS2000® based computers), MACINTOSH®, or UNIX® based (e.g., SUN® work station) computers.
One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
VIII. Administration and Pharmaceutical Compositions
Modulators of the polynucleotides or polypeptides of the invention (e.g., antagonists or agonists) can be administered directly to a mammalian subject for modulation of activity of those molecules in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Diseases that can be treated include the following, which include the corresponding reference number from Morrison, DSM-IV Made Easy, 1995: Schizophrenia, Catatonic, Subchronic, (295.21); Schizophrenia, Catatonic, Chronic (295.22); Schizophrenia, Catatonic, Subchronic with Acute Exacerbation (295.23); Schizophrenia, Catatonic, Chronic with Acute Exacerbation (295.24); Schizophrenia, Catatonic, in Remission (295.55); Schizophrenia, Catatonic, Unspecified (295.20); Schizophrenia, Disorganized, Subchronic (295.11); Schizophrenia, Disorganized, Chronic (295.12); Schizophrenia, Disorganized, Subchronic with Acute Exacerbation (295.13); Schizophrenia, Disorganized, Chronic with Acute Exacerbation (295.14); Schizophrenia, Disorganized, in Remission (295.15); Schizophrenia, Disorganized, Unspecified (295.10); Schizophrenia, Paranoid, Subchronic (295.31); Schizophrenia, Paranoid, Chronic (295.32); Schizophrenia, Paranoid, Subchronic with Acute Exacerbation (295.33); Schizophrenia, Paranoid, Chronic with Acute Exacerbation (295.34); Schizophrenia, Paranoid, in Remission (295.35); Schizophrenia, Paranoid, Unspecified (295.30); Schizophrenia, Undifferentiated, Subchronic (295.91); Schizophrenia, Undifferentiated, Chronic (295.92); Schizophrenia, Undifferentiated, Subchronic with Acute Exacerbation (295.93); Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation (295.94); Schizophrenia, Undifferentiated, in Remission (295.95); Schizophrenia, Undifferentiated, Unspecified (295.90); Schizophrenia, Residual, Subchronic (295.61); Schizophrenia, Residual, Chronic (295.62); Schizophrenia, Residual, Subchronic with Acute Exacerbation (295.63); Schizophrenia, Residual, Chronic with Acute Exacerbation (295.94); Schizophrenia, Residual, in Remission (295.65); Schizophrenia, Residual, Unspecified (295.60); Delusional (Paranoid) Disorder (297.10); Brief Reactive Psychosis (298.80); Schizophreniform Disorder (295.40); Schizoaffective Disorder (295.70); Induced Psychotic Disorder (297.30); Psychotic Disorder NOS (Atypical Psychosis) (298.90); Personality Disorders, Paranoid (301.00); Personality Disorders, Schizoid (301.20); Personality Disorders, Schizotypal (301.22); Personality Disorders, Antisocial (301.70); Personality Disorders, Borderline (301.83) and bipolar disorders, maniac, hypomaniac, dysthymic or cyclothymic disorders, substance-induced mood disorders, major depression, psychosis, including paranoid psychosis, catatonic psychosis, delusional psychosis, having schizoaffective disorder, and substance-induced psychotic disorder.
In some embodiments, modulators of polynucleotides or polypeptides of the invention can be combined with other drugs useful for treating mental disorders including useful for treating mood disorders, e.g., schizophrenia, bipolar disorders, or major depression. In some preferred embodiments, pharmaceutical compositions of the invention comprise a modulator of a polypeptide of polynucleotide of the invention combined with at least one of the compounds useful for treating schizophrenia, bipolar disorder, or major depression, e.g., such as those described in U.S. Pat. Nos. 6,297,262; 6,284,760; 6,284,771; 6,232,326; 6,187,752; 6,117,890; 6,239,162 or 6,166,008.
The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)).
The modulators (e.g., agonists or antagonists) of the expression or activity of the a polypeptide or polynucleotide of the invention, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation or in compositions useful for injection. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.
The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial response in the subject over time. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the mental disorder. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular compound or vector in a particular subject.
In determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies. In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
For administration, modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
IX. Gene Therapy Applications
A variety of human diseases can be treated by therapeutic approaches that involve stably introducing a gene into a human cell such that the gene is transcribed and the gene product is produced in the cell. Diseases amenable to treatment by this approach include inherited diseases, including those in which the defect is in a single or multiple genes. Gene therapy is also useful for treatment of acquired diseases and other conditions. For discussions on the application of gene therapy towards the treatment of genetic as well as acquired diseases, see, Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932 (1993).
In the context of the present invention, gene therapy can be used for treating a variety of disorders and/or diseases in which the polynucleotides and polypeptides of the invention has been implicated. For example, compounds, including polynucleotides, can be identified by the methods of the present invention as effective in treating a mental disorder. Introduction by gene therapy of these polynucleotides can then be used to treat, e.g., mental disorders including mood disorders and psychotic disorders.
A. Vectors for Gene Delivery
For delivery to a cell or organism, the polynucleotides of the invention can be incorporated into a vector. Examples of vectors used for such purposes include expression plasmids capable of directing the expression of the nucleic acids in the target cell. In other instances, the vector is a viral vector system wherein the nucleic acids are incorporated into a viral genome that is capable of transfecting the target cell. In a preferred embodiment, the polynucleotides can be operably linked to expression and control sequences that can direct expression of the gene in the desired target host cells. Thus, one can achieve expression of the nucleic acid under appropriate conditions in the target cell.
B. Gene Delivery Systems
Viral vector systems useful in the expression of the nucleic acids include, for example, naturally occurring or recombinant viral vector systems. Depending upon the particular application, suitable viral vectors include replication competent, replication deficient, and conditionally replicating viral vectors. For example, viral vectors can be derived from the genome of human or bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated virus, minute virus of mice (MVM), HIV, sindbis virus, and retroviruses (including but not limited to Rous sarcoma virus), and MoMLV. Typically, the genes of interest are inserted into such vectors to allow packaging of the gene construct, typically with accompanying viral DNA, followed by infection of a sensitive host cell and expression of the gene of interest.
As used herein, “gene delivery system” refers to any means for the delivery of a nucleic acid of the invention to a target cell. In some embodiments of the invention, nucleic acids are conjugated to a cell receptor ligand for facilitated uptake (e.g., invagination of coated pits and internalization of the endosome) through an appropriate linking moiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180). For example, nucleic acids can be linked through a polylysine moiety to asialo-oromucocid, which is a ligand for the asialoglycoprotein receptor of hepatocytes.
Similarly, viral envelopes used for packaging gene constructs that include the nucleic acids of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells (see, e.g., WO 93/20221, WO 93/14188, and WO 94/06923). In some embodiments of the invention, the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis (Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). In other embodiments, molecular conjugates of the instant invention can include microtubule inhibitors (WO/9406922), synthetic peptides mimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem. 269:12918-12924 (1994)), and nuclear localization signals such as SV40 T antigen (WO93/19768).
Retroviral vectors are also useful for introducing the nucleic acids of the invention into target cells or organisms. Retroviral vectors are produced by genetically manipulating retroviruses. The viral genome of retroviruses is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy which is integrated into the chromosomal DNA of transduced cells with a high degree of stability and efficiency. The integrated DNA copy is referred to as a provirus and is inherited by daughter cells as is any other gene. The wild type retroviral genome and the proviral DNA have three genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5′ and 3′ LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site) (see, Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984)).
The design of retroviral vectors is well known to those of ordinary skill in the art. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis-acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well known in the art and are used to construct retroviral vectors. Preparation of retroviral vectors and their uses are described in many publications including, e.g., European Patent Application EPA 0 178 220; U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504-512 (1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988); Miller et al. Biotechniques 7:981-990 (1989); Miller (1992) supra; Mulligan (1993), supra; and WO 92/07943.
The retroviral vector particles are prepared by recombinantly inserting the desired nucleotide sequence into a retrovirus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line. The resultant retroviral vector particle is incapable of replication in the host cell but is capable of integrating into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, the patient is capable of producing, for example, a polypeptide or polynucleotide of the invention and thus restore the cells to a normal phenotype.
Packaging cell lines that are used to prepare the retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging, but which are incapable of producing infectious virions. The defective retroviral vectors that are used, on the other hand, lack these structural genes but encode the remaining proteins necessary for packaging. To prepare a packaging cell line, one can construct an infectious clone of a desired retrovirus in which the packaging site has been deleted. Cells comprising this construct will express all structural viral proteins, but the introduced DNA will be incapable of being packaged. Alternatively, packaging cell lines can be produced by transforming a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.
A number of packaging cell lines suitable for the present invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al., J. Virol. 65:2220-2224 (1991)). Examples of other packaging cell lines are described in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller (1990), supra.
Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins may be used. Alternatively, amphotropic or xenotropic envelope proteins, such as those produced by PA317 and GPX packaging cell lines may be used to package the retroviral vectors.
In some embodiments of the invention, an antisense polynucleotide is administered which hybridizes to a gene encoding a polypeptide of the invention. The antisense polypeptide can be provided as an antisense oligonucleotide (see, e.g., Murayama et al., Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Genes encoding an antisense nucleic acid can also be provided; such genes can be introduced into cells by methods known to those of skill in the art. For example, one can introduce an antisense nucleotide sequence in a viral vector, such as, for example, in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997)), in adeno-associated virus (see, e.g., Xiao et al., Brain Res. 756:76-83 (1997)), or in other systems including, but not limited, to an HVJ (Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et al., Ann. NY Acad. Sci. 811:299-308 (1997)), a “peptide vector” (see, e.g., Vidal et al., CR Acad. Sci III 32:279-287 (1997)), as a gene in an episomal or plasmid vector (see, e.g., Cooper et al., Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584 (1997)), as a gene in a peptide-DNA aggregate (see, e.g., Niidome et al., J. Biol. Chem. 272:15307-15312 (1997)), as “naked DNA” (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466), in lipidic vector systems (see, e.g., Lee et al., Crit Rev Ther Drug Carrier Syst. 14:173-206 (1997)), polymer coated liposomes (U.S. Pat. Nos. 5,213,804 and 5,013,556), cationic liposomes (Epand et al., U.S. Pat. Nos. 5,283,185; 5,578,475; 5,279,833; and 5,334,761), gas filled microspheres (U.S. Pat. No. 5,542,935), ligand-targeted encapsulated macromolecules (U.S. Pat. Nos. 5,108,921; 5,521,291; 5,554,386; and 5,166,320).
Upregulated transcripts listed in the biomarker tables herein which are correlated with mental disorders may be targeted with one or more short interfering RNA (siRNA) sequences that hybridize to specific sequences in the target, as described above. Targeting of certain brain transcripts with siRNA in vivo has been reported, for example, by Zhang et al., J. Gene. Med., 12:1039-45 (2003), who utilized monoclonal antibodies against the transferrin receptor to facilitate passage of liposome-encapsulated siRNA molecules through the blood brain barrier. Targeted siRNAs represent useful therapeutic compounds for attenuating the over-expressed transcripts that are associated with disease states, e.g., MDD, BP, and other mental disorders.
In another embodiment, conditional expression systems, such as those typified by the tet-regulated systems and the RU-486 system, can be used (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These systems impart small molecule control on the expression of the target gene(s) of interest.
In another embodiment, stem cells engineered to express a transcript of interest can implanted into the brain.
C. Pharmaceutical Formulations
When used for pharmaceutical purposes, the vectors used for gene therapy are formulated in a suitable buffer, which can be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al. Biochemistry 5:467 (1966).
The compositions can additionally include a stabilizer, enhancer, or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the nucleic acids of the invention and any associated vector. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).
D. Administration of Formulations
The formulations of the invention can be delivered to any tissue or organ using any delivery method known to the ordinarily skilled artisan. In some embodiments of the invention, the nucleic acids of the invention are formulated in mucosal, topical, and/or buccal formulations, particularly mucoadhesive gel and topical gel formulations. Exemplary permeation enhancing compositions, polymer matrices, and mucoadhesive gel preparations for transdermal delivery are disclosed in U.S. Pat. No. 5,346,701.
E. Methods of Treatment
The gene therapy formulations of the invention are typically administered to a cell. The cell can be provided as part of a tissue, such as an epithelial membrane, or as an isolated cell, such as in tissue culture. The cell can be provided in vivo, ex vivo, or in vitro.
The formulations can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods. In some embodiments of the invention, the nucleic acids of the invention are introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics. In further embodiments, the nucleic acids are taken up directly by the tissue of interest.
In some embodiments of the invention, the nucleic acids of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).
X. Diagnosis of Mood Disorders and Psychotic Disorders
The present invention also provides methods of diagnosing mood disorders (such as major depression or bipolar disorder), psychotic disorders (such as schizophrenia), or a predisposition of at least some of the pathologies of such disorders. Diagnosis involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy person not suffering from a mood disorder or a psychotic disorder or under the effects of medication or other drugs. Variation of levels of a polypeptide or polynucleotide of the invention from the baseline range (either up or down) indicates that the patient has a mood disorder or a psychotic disorder or at risk of developing at least some aspects of a mood disorder or a psychotic disorder. In some embodiments, the level of a polypeptide or polynucleotide of the invention are measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein.
Antibodies can be used in assays to detect differential protein expression in patient samples, e.g., ELISA assays, immunoprecipitation assays, and immunohistochemical assays. PCR assays can be used to detect expression levels of nucleic acids, as well as to discriminate between variants in genomic structure, such as insertion/deletion mutations (e.g., PSPHL).
In the case where absence of gene expression is associated with a disorder, the genomic structure of a gene such as PSPHL can be evaluated with known methods such as PCR to detect deletion or insertion mutations associated with disease suspectibility. Conversely, the presence of mRNA or protein corresponding to the PSPHL gene would indicate that an individual does not have the PSPHL deletion associated with susceptibility to BP. Thus, diagnosis can be made by detecting the presence or absence of mRNA or protein, or by examining the genomic structure of the gene. Any combination of exons or non-transcribed regions can be used to detect the deletion allele. For example, the presence of exon 4 but not exons 1, 2, and/or 3 would indicate the presence of the deletion allele. Similarly, deletion of the promoter region would indicate the deletion allele. Any significant mRNA detection, especially detection of an mRNA comprising exons 1, 2, and/or 3, would indicate the absence of the deletion allele, which is not transcribed due to the promoter deletion.
Single nucleotide polymorphism (SNP) analysis is also useful for detecting differences between alleles of the polynucleotides (e.g., genes) of the invention. SNPs linked to genes encoding polypeptides of the invention are useful, for instance, for diagnosis of diseases (e.g., mood disorders such as bipolar disease, major depression, and schizophrenia disorders) whose occurrence is linked to the gene sequences of the invention. For example, if an individual carries at least one SNP linked to a disease-associated allele of the gene sequences of the invention, the individual is likely predisposed for one or more of those diseases. If the individual is homozygous for a disease-linked SNP, the individual is particularly predisposed for occurrence of that disease. In some embodiments, the SNP associated with the gene sequences of the invention is located within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 base pairs from the gene sequence.
Various real-time PCR methods can be used to detect SNPs, including, e.g., Taqman or molecular beacon-based assays (e.g., U.S. Pat. Nos. 5,210,015; 5,487,972; Tyagi et al., Nature Biotechnology 14:303 (1996); and PCT WO 95/13399 are useful to monitor for the presence of absence of a SNP. Additional SNP detection methods include, e.g., DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis, or are described in, e.g., U.S. Pat. No. 6,177,249; Landegren et al., Genome Research, 8:769-776 (1998); Botstein et al., Am J Human Genetics 32:314-331 (1980); Meyers et al., Methods in Enzymology 155:501-527 (1987); Keen et al., Trends in Genetics 7:5 (1991); Myers et al., Science 230:1242-1246 (1985); and Kwok et al., Genomics 23:138-144 (1994). PCR methods can also be used to detect deletion/insertion polymorphisms, such as the deletion polymorphism of the PSPHL gene associated with suspectibility to BP.
In some embodiments, the level of the enzymatic product of a polypeptide or polynucleotide of the invention is measured and compared to a baseline value of a healthy person or persons. Modulated levels of the product compared to the baseline indicates that the patient has a mood disorder or a psychotic disorder or is at risk of developing at least some aspects of a mood disorder or a psychotic disorder. Patient samples, for example, can be blood, urine or tissue samples.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
EXAMPLES
Example 1
Identification of FGF Pathway Genes Dysregulated in MDD
Major depressive disorder (MDD) and bipolar disorder (BP) are affective diseases that strike a significant proportion of the population. These complex genetic disorders arise from the interplay of vulnerability genes and environmental stressors, impacting neural circuits that control mood. Beyond the role of limbic structures, mood disorders are hypothesized to involve aberrant activity of the cerebral cortex. Thus, imaging techniques have implicated the dorsolateral prefrontal (DLPFC) and anterior cingulate (AnCg) cortices in mood disorders since affected subjects display changes in volumetric measurements (Harrison, P. J. Brain 125, 1428-49 (2002).) and altered activity in response to a cognitive challenge. (Kruger, S., Seminowicz, D., Goldapple, K., Kennedy, S. H. & Mayberg, H. S. Biol Psychiatry 54, 1274-83 (2003)) Using a candidate approach, studies have demonstrated altered cortical expression of specific neurotransmitter- and stress-related genes in affective illness. However, the full extent of the alteration in cortical activity had not been described, nor had an unbiased “discovery” approach been applied to characterize it.
We have applied microarray technology to the study of DLPFC and AnCg in post-mortem samples from MDD, BP and non-psychiatric controls. This represents the first transcriptional profiling study demonstrating significant alterations of gene expression in major depression, and the first one contrasting the two major mood disorders (MDD and BP) with the same group of controls. Here we report the dysregulation of fibroblast growth factor (FGF) system transcripts specifically in MDD.
Human Studies
The studies were carried out in a carefully selected cohort of postmortem human brains, and replicated in a separate cohort (see Table 1a and 1b). The Affymetrix HG-133A array contains probe sets for 21 FGF system transcripts, including all 4 receptors (FGFR1, 2, 3, 4) and 12 FGF peptide ligands (FGF1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 17, 18, 20, 21, 22, 23). Of these only 10 were reliably detected in the regions assayed and include three of the FGF receptors (FGFR1, 2, and 3) and seven FGF ligands (FGF1, 2, 7, 9, 12, 13,14). Of the ten FGF transcripts reliably detected, seven were significantly altered in one of the two regions studied—four were significantly differentially expressed in the DLPFC of MDD subjects, including 2 FGF receptors (FGFR2 and 3) and 2 FGF ligands (FGF1 and 9); and six were significantly differentially expressed in the AnCg including two receptors (FGFR2 and 3) and four ligands (FGF1, 2, 9 and 12). The probability that this family of molecules would have emerged by chance, based on a hypergeometric distribution, is p<0.001. These data are summarized in Table 2, which also reports on the transcripts confirmed by real-time PCR analysis and/or those replicated in a second independent cohort of MDD and control subjects. Importantly, none of the above transcripts were observed to be differentially expressed in BP by microarray in either cohort or by real-time PCR analysis, demonstrating the specificity of this dysregulation for MDD.
Given this selective dysregulation of FGF system transcripts in MDD we asked whether these changes might be secondary to antidepressant therapy since a subset of the subjects was on antidepressants, and the majority of those were on specific serotonin reuptake inhibitors (SSRI's). Thus, we separated our microarray data into MDD subjects prescribed SSRI's (n=5) and those not prescribed SSRI's (n=4) for statistical comparisons. This analysis showed that FGFR3 and FGF2 had a tendency toward up-regulation (p=0.15 and 0.07, respectively) and that FGF 9 showed a tendency toward down-regulation (p=0.12) by SSRI treatment, all opposing the directionality of dysregulation we see in MDD subjects relative to controls. No other FGF system transcripts approached significant differences in this analysis. These data strongly suggest that our observations are not secondary to SSRI treatment. Furthermore, the observed attenuation of FGF transcript dysregulation in the SSRI prescribed group suggests that the normalization of FGF system might be one mechanism of action of this class of drugs since several FGF transcripts appear to be altered in severe depression and are partially reversed by SSRI therapy.
Rodent Anatomical Studies; Effect of Fluoxetine.
We studied the anatomical expression of FGFR2 in rats subjected to chronic fluoxetine treatment relative to controls (FIG. 1). FGFR2 mRNA expression was quantified in the retrosplenial cortex and in the hippocampus since this area has recently been implicated in the mode of action of antidepressants (Santarelli, L. et al. Science 301, 805-9 (2003)). Results show that FGFR2 message is significantly increased by fluoxetine in all hippocampal subfields (CA1, CA2, CA3, and dentate gyrus) and the retrosplenial cortex (Fisher's PLSD, p=0.0049).
The implication of the FGF system in MDD has emerged from our studies utilizing microarray technology to study human psychiatric illness. Other growth factors have been hypothesized to contribute to the etiology and maintenance of such illnesses and can offer a framework in which to place our own findings. Most notably, brain derived neurotrophic factor (BDNF) has been repeatedly implicated in MDD, BP and in SZ. BDNF mRNA levels are reportedly decreased in the DLPFC (Weickert, C. S., et al., Mol Psychiatry 8, 592-610 (2003)) of schizophrenics and BDNF protein levels are decreased in serum of MDD (Shimizu, E. et al. Biol Psychiatry 54, 70-5 (2003)) patients. Furthermore, BDNF expression is regulated by antipsychotic (Chlan-Fourney, J. et al., Brain Res 954, 11-20 (2002)) and antidepressant drugs (Shimizu, E. et al. Biol Psychiatry 54, 70-5 (2003)), (Dias, B. G., et al., Neuropharmacology 45, 553-63 (2003)). A smaller volume of literature implicates other growth factors, including nerve growth factor (Parikh, V., Evans, D. R., Khan, M. M. & Mahadik, S. P. Schizophr Res 60, 117-23 (2003)), epidermal growth factor (Futamura, T. et al. Mol Psychiatry 7, 673-82 (2002)) and neurotrophin-3 (Hock, C. et al. Mol Psychiatry 5, 510-3 (2000)) in psychiatric illness.
Growth factors play significant roles in development and maintenance of the central nervous system. In the developing brain, they are involved in specific neuronal terminal differentiation and migration to appropriate subfields. In the adult brain they are critical in neuronal survival, axonal branching and synaptic plasticity. Specifically, FGF2 (Viti, J., Gulacsi, A. & Lillien, L. J Neurosci 23, 5919-27 (2003)) and FGF8 (Gunhaga, L. et al. Nat Neurosci 6, 701-7 (2003)) have been shown to interact with Wnt in the development of the cortex in mouse and chick embryos, respectively. In the adult brain, FGF2 promotes neuronal survival and axonal branching (Abe, K. & Saito, H. Pharmacol Res 43, 307-12 (2001)) and its expression is modulated by stress (Molteni, R. et al., Brain Res Rev 37, 249-58 (2001)).
Together, these results lead to the novel hypothesis that dysregulation of the FGF system contributes to either the vulnerability to MDD or the expression of the illness, and that antidepressants might attenuate this dysregulation. Animal models will be crucial for defining the involvement of the FGF system in emotionality and elucidating its role in neural plasticity and antidepressant action.
Example 2
Identification of Novel Insertion/Deletion Polymorphism in PSPHL Gene and Association of Deletion Mutation with BP Susceptibility
Evaluation of PSPHL expression in anterior cingulated cortex by quantitative RT-PCR reveals that PSPHL shows a dichotomous (present or absent) pattern of expression among individuals. In our first cohort, none of the 9 BPD patients (0%) shows PSPHL expression, while 7 out of 11 MDD patients (64%) and 8 out of 20 controls (40%) show sufficient expression of PSPHL. The probability of distribution of the present/absent expression pattern between BPD and controls is 0.018, and between MDD and controls is 0.105 based on the Fisher exact test. To assure this significant contrast between BPD and controls, we measured PSPHL expression in an additional 9 BPD and 20 control individuals (for a total of 18 BPD and 40 controls) using quantitative RT-PCR. In this larger sample set, none of the 18 BPD patients (0%) shows any expression of PSPHL, while 16 out of 40 controls (40%) show sufficient expression of the transcript (p value=0.0008).
The fact that PSPHL shows dichotomous present/absent pattern of expression among individuals with brain-wide consistency suggests genetic variation in its regulation. Since genomic organization of PSPHL has not been characterized (Planitzer, supra (1998)), we have identified genomic organization of PSPHL as shown in FIG. 14. The PSPHL gene consists of 4 exons. Exons 1, 2, 3 and 4 are 213 bp, 114 bp, 122 bp and 501 bp, in length, respectively, and span introns 1, 2 and 3 (3221bp, 829 bp and 11939 bp, in length, respectively). Further, the PSPHL gene has two alternative transcripts, one of which utilizes the exons 1-4 (PSPHL-A in FIG. 14), while another utilizes the exons 1, 2 and 4 (PSPHL-B). We have also identified an insertion/deletion polymorphism at the PSPHL locus. The deleted genomic region spans more than 30 kb, including the promoter region and the exons 1, 2 and 3 of PSPHL gene. This genetic variance explains the present/absent pattern of the PSPHL expression. An over-representation of the deletion allele resulting in the absence of PSPHL expression increases susceptibility to BPD.
PSPHL and PSPH are highly homologous, but appear to be different genes, which are about 2OOkb apart from each other on chromosome 7p11.2 region. Especially, exons 2-4 of PSPHL are highly homologous to exons 4 and 8 of PSPH gene. Predicted amino acid sequences of PSPH, PSPHL-A and PSPHL-B are shown in FIG. 15. PSPHL-A and PSPHL-B share N-terminal 57 common amino acids, transcribed from exons 1 and 2. PSPHL-A has unique C-terminal 36 amino acids, transcribed from exon3, while PSPHL-B has unique C-terminal 17 amino acids, transcribed from exon 4. PSPH and PSPHL-A&B have 31 amino acids in common. The common amino acids locates at the N-terminal end of PSPH and middle region (25th-56th amino acids) of PSPHL-A and B. The common region contains consensus phosphorylation site of Na/K ATPase and casein kinase II phospholyration site. Based on the similarity in the structure, PSPHL shares some function with PSPH gene.
PSPH is the rate limiting enzyme for serine synthesis. PSPH has haloacid dehalogenase-like hydrolase domain, which is responsible for the activity. Greater than 90% of L-serine in brain is formed via the phosphorylated pathway. PSPH may be dimeric from of the enzyme with a monomeric molecular weight of 26 kDa. L-serine is converted to sphingomyelins and gangliosides, as well as L-glycine and D-serine, both of which act as coagonist for NMDA receptor associated glycine binding site. L-glycine is also an agonist for strychnine-sensitive glycine receptor (FIG. 16). PSPHL is involved in serine amino acid metabolic pathway, and may involved in other pathways as well.
Example 3
Post-Natal Injection of FGF2
This Example shows that neonatal administration of FGF-2 affects long-term alterations in hippocampal volume, emotional reactivity and learning and memory. Sprague-Dawley rats were injected with either vehicle or FGF-2 (20 ng/g, s.c.) on postnatal day 2 (PD2). Three weeks after injection we evaluated dentate gyrus volume and cell counts by Nissl staining. We also assessed neurogenesis by BrdU and Ki-67 immunohistochemistry at the 23 day time point. In adult rats, we tested locomotor activity, anxiety behavior and learning and memory. The animals were sacrificed, and the brains collected for in situ hybridization (FGF markers, stress markers). Results to date have shown the following: FGF-2 injected rats exhibited a 10.5% increase in dentate gyrus volume. The results show that FGF-2 significantly increased locomotor activity over controls in a novel environment. Increased activity in response to novelty has been associated with a host of other measures including decreased anxiety-like behavior. Furthermore, adult rats that received FGF-2 as neonates also performed significantly better than controls in the Morris water maze.
Example 4
FGF2 Expression
While evidence has linked growth factors such as BDNF to environmental complexity (EC), responsiveness to stress, and antidepressant action, few studies focused on the role of the FGF system in emotional reactivity. Recent data from our laboratory suggest that a single postnatal injection of FGF-2 significantly alters locomotor activity in response to a novel environment (Turner et al., SFN abstracts 2004). Since increased responsiveness to novelty is associated with decreased anxiety-like behavior, we propose that FGF-2 may be correlated with other indices of emotionality. We tested the hypothesis that changes in emotionality associated with EC may be related to FGF-2 gene expression in the hippocampus. Young adult male Sprague-Dawley rats were either exposed to a complex environment for 21 days or to standard cages. Following this treatment, rats were returned to standard cages for two weeks. Brains were then processed for neurogenesis by BrdU and Ki-67 immunohistochemistry. Another group of rats was tested in the elevated plus-maze (EPM) and then sacrificed for in situ hybridization. Compared to controls, EC rats showed significantly less anxiety-like behavior in the EPM and exhibited a 23% increase in FGF-2 expression in the hippocampus. There was a significant positive correlation between FGF-2 mRNA levels in hippocampal CA2 and time spent in the open arms of the EPM. Whether these results relate to levels of neurogenesis in the hippocampus is currently being determined. These findings are consistent with our observations in human postmortem brains (Evans et al, SfN Abstracts 2004) showing that expression of several members of the FGF family is decreased in major depression. Together, these findings implicate the FGF system in emotionality and mood disorders.
Example 5
Cyclic Amp Signaling Pathway Genes Differentially Expressed in BPD and/or MDD Patients
Two independent cohorts A and B were analyzed separately in this study. Cohort A consisted of 22 subjects including 7 healthy control subjects, 6 patients with BPD and 9 patients with MDD. Cohort B consisted of 12 subjects including 5 MDD and 7 controls. All subjects in this study did not have specific agonal conditions including hypoxia, coma, pyrexia, seizure, dehydration, hypoglycemia, multi-organ failure, skull fracture, ingestion of neurotoxic substances or prolonged agonal duration, which is known to affect tissue pH, RNA integrity and gene expression profile in postmortem brain, and showed brain tissue pH of more than 6.5. In order to detect reliable gene expression differences between diagnostic groups, we performed experimentally as well as biologically replicated experiments as follows. Experiment 1: Total RNA was extracted from AnCg, DLPFC and CB of the Cohort A, and purified with silica-based mini-spin columns (Qiagen RNeasy Mini Kit, Valencia, Calif.). The oligonucleotide microarray experiments were carried out following the manufacturer's protocol (Affymetrix, Santa Clara, Calif.). For technical replication, each of RNA samples was run on Affymetrix U95Av2 GeneChips at two laboratories. Experiment 2: For further technical replication, samples from the 22 subjects from cohort A were reanalyzed in AnCg and DLPFC utilizing U133A GeneChips at two laboratories. Experiment 3: Samples from the additional cohort B were analyzed on U133A GeneChips in AnCg and DLPFC at two laboratories. Signal intensity data was extracted with Robust Multi-array Average (RMA) for each probe set and each subject. Gene-wise Pearson's correlation coefficients between experimental duplicates were calculated, and only the genes significantly correlated between experimental duplicates were considered to be reliably detectable genes, and subjected to the downstream analyses. For these reliably detectable genes, mixed-model multivariate ANOVA analyses were employed utilizing Partek Pro 6.0 (Partek, St.Charles, Mo.) to adjust the effect of the diagnostic classification (BPD, MDD, control) for possible confounders, including site for experiment, experimental batch, and gender. Post hoc tests (least-squares difference) were run to generate p value for the differences between case and control means, and false discovery rate multiple comparison corrections at the level of accepting 5% false positives was applied to each ANOVA result. When the p value passed false discovery rate multiple testing correction at the level of accepting 5% false positives and percent fold change exceeds 20%, the genes were consider to be significantly differentially expressed between case and control groups. Also, in order to evaluate more subtle but consistent expression differences between case and control groups, we also selected genes passed p value of 0.05 regardless of FDR correction and % FC>10% on both experimental duplicates utilizing U95Av2 or U133A GeneChips.
FIG. 26A and Table 14 summarize cAMP signaling pathway related genes which were differentially expressed in anterior cingulate cortex (AnCg) of bipolar disorder (BPD) patients compared with controls. Among GPCRs coupled with G protein inhibitory subunit (Gi), which inhibits adenylate cyclase activity, neuropeptide Y receptor 1 (NPYR1) was significantly increased in AnCg of BPD. The ligand, neuropeptide Y was also significantly increased in AnCg of BPD. Gi-linked metabotropic glutamate receptor 3 (GRM3) was also increased in AnCg as well as dorsolateral prefrontal cortex (DLPFC) of BPD. Somatostatin (SST), a ligand for Gi-coupled GPCR, was significantly increased in AnCg in our microarray data.
Contrasted to the finding in AnCg, SST mRNA expression was decreased in DLPFC of BPD as well as major depressive disorder (MDD). Adrenergic beta-1 receptor (ADRB1) was decreased by 13-18% in DLPFC of BPD, although the change did not reach the significant criteria (Table 17). Proenkephalin (PENK), a ligand for Gi-coupled GPCR, was not altered in AnCg and DLPFC of BPD and MDD, but significantly increased in CB of both BPD and MDD.
Messenger RNA expression level of G protein alpha subunit inhibitory peptide 1 (GNAI1) and phosphodiesterase 1A (PDE1A) were significantly increased in AnCg of BPD patients. Protein kinase A inhibitor alpha (PKIA) and cyclin dependent kinase 5 (CDK5), phosphodiesterase 8A (PDE8A) and protein phosphatase 1, catalytic subunit, alpha (PPP1CA) did not reach significant criteria, but were increased by 10%-20% in AnCg of BPD. Thus, mRNA expression of molecules suppressing cAMP concentration and PKA activity were generally increased in BPD, while molecules activating cAMP signaling (Gs-coupled GPCR, Gs, adenylate cyclase, protein kinase A) did not show significant alteration at the transcript level.
FIG. 26 and Table 14 summarize cAMP signaling pathway related genes which were differentially expressed in AnCg of MDD patients compared with controls. Gi-linked endothelial differentiation GPCR 1 (EDG1) was significantly decreased in AnCg of MDD. Regulator of G protein signaling 20 (RGS20), phosphodiesterase 8A (PDE8A), and protein phosphatase 1 regulatory subunit 3C (PPPlR3C) showed significantly lower expression in AnCg of MDD patients compared with controls. Expression levels of PDE8A and PPP1R3C mRNAs were significantly lower also in DLPFC of MDD (Table 15). Significant decrease in RGS20 expression in MDD was observed also in CB (Table 16). Thus, mRNA expression of molecules suppressing cAMP concentration and PKA activity were generally decreased in MDD, while the molecules activating cAMP signaling did not show significant alteration at the transcript level.
Phosphatidylinositol Signaling Pathway
FIG. 26C and Table 14 summarize phosphatidylinositol signaling (PI) pathway related genes which were differentially expressed in AnCg of BPD patients compared with controls. Gq-linked tachykinin (neuropeptide K) receptor 2 (TACR2) was significantly decreased in AnCg of BPD. Messenger RNA expression of inositol polyphosphate-1-phosphatase (INPP1) was significantly higher, while CDP-diacylglycerol synthase 1 (CDS 1), a regulatory subunit of class I phosphatidylinositol 3 kinase (PIK3R1) and protein kinase C iota (PKCI) were significantly lower in AnCg of BPD patients compared with control group. Inositol 1,4,5-trisphosphate 3-kinase B (ITPKB) and catalytic beta subunit of class II phosphatidylinositol 3 kinase (PIK3C2B) did not reach significance criteria, but increased by 10%-20% in AnCg of BPD.
FIG. 26D and Table 14 summarize PI signaling pathway related genes which were differentially expressed in AnCg of MDD patients compared with controls. Gq-linked neurotensin receptor 2 (NTSR2) and endothelin receptor type B (EDNRB) were significantly decreased in AnCg of MDD. Messenger RNA expression of inositol polyphosphate-5-phosphatase F (INPP5F) was significantly higher, while inositol 1,4,5-trisphosphate 3-kinase B (ITPKB) and catalytic alpha subunit of class II phosphatidylinositol 3 kinase (PIK3C2A) were significantly lower in AnCg of MDD compared with control. Inositol polyphosphate-5-phosphatase A (INPP5A), protein kinase C beta 1 (PKCB 1) and inositol 1,4,5-triphosphate receptor type 1 (ITPR1) did not reach the significance criteria, but increased by 10-20% in AnCg of MDD patients. Significant decrease in ITPKB mRNA expression was observed also in DLPFC of MDD (Table 15). Neither G protein alpha q subunit nor phospholipase C beta mRNAs were altered in any of the brain regions of the disorder groups.
Other G Protein-Coupled Receptors
Among all GPCRs, the most consistent differential expression patterns throughout our experiments were observed in G protein-coupled receptor family C, group 5, member B (GPRC5B) and G protein-coupled receptor 37 (GPR37). GPRC5B was significantly increased in AnCg and DLPFC of BPD. GPRC5B was significantly decreased in AnCg, DLPFC and CB of MDD patients. A significant decrease of GPRC5B in AnCg and DLPFC of MDD patients was replicated by the experiments utilizing another independent cohort B. GPR37 was also significantly increased in AnCg of BPD, and significantly decreased in AnCg, DLPFC and CB of MDD.
Quantitative RT-PCR
For further technical evaluation of the microarray data, we evaluated mRNA expression levels by real-time quantitative reverse transcriptase PCR (qRT-PCR) for the following 7 genes, in anterior cingulate cortex (AnCg): Somatostatin (SST), neuropeptide Y (NPY), G protein-coupled receptor C-5-B (GPRC5B), G protein-coupled receptor 37 (GPR37), regulator of G-protein signaling 20 (RGS20), inositol polyphosphate-1-phosphatase (INPP1) and protein phosphatase 1 regulatory subunit 3C(PPPIR3C).
In consistent with microarray data, qRT-PCR data showed that mRNA expressions of neuropeptide Y (NPY), G protein-coupled receptor C-5-B (GPRC5B), G protein-coupled receptor 37 (GPR37), inositol polyphosphate-1-phosphatase (INPP1) were significantly increased in AnCg of BPD, and expression level of GPRC5B, GPR37, regulator of G-protein signaling 20 (RGS20) and protein phosphatase 1 regulatory subunit 3C (PPP1R3C) were significantly decreased in the AnCg of MDD group. While somatostatin (SST) mRNA expression was increased in AnCg of BPD in both microarray experimental duplicates utilizing U95Av2 and U133A, the finding was not replicated by qRT-PCR (Table 17).
In Situ Hybridization
We performed in situ hybridization for GPR37 using. GPR37 mRNA is preferentially expressed in subcortical white matter. GPR37 expression in the deeper layers (V-VI) is relatively higher than the superficial layers (I-III). GPR37 mRNA expression in subcortical white matter was higher in AnCg of the BPD subjects compared to the control subjects. GPR37 mRNA expression was rarely detected in AnCg of the MDD subjects analyzed. FIG. 27 shows the dysregulation of genes involved in cAMP- and phosphatidylinositol signaling pathways in brain tissue from patients with BPD and MDD.
Gene Expression Changes in Amygdala, Hippocampus, Nucleus Accumbens of Bipolar Disorder and Major Depressive Disorder.
We applied the same experimental design on amygdala, hippocampus and nucleus accumbens of BPD and MDD. Table 18 summarizes genes which are differentially expressed in amygdala, hippocampus and nucleus accumbens of BPD patients. Table 19 summarizes genes differentially expressed in the three brain regions of MDD.
Example 6
This Example shows gene dysregulation in pathways related to Mitochondria, Proteasome, Apoptosis, and Chaperone in Mood Disorder. Three brain regions were studied: AnCg, Cerebellum, and DLPFC. The results are compiled in Tables 20-22.
Example 7
NCAM1 Association with Bipolar Disorder and Schizophrenia and Splice Variants of NCAM1
SNP and sample selection: Genomic DNA (gDNA) was extracted from human postmortem brain cerebellum tissue. Primers were designed for SNP 9 and then tested via PCR to determine correct band size. Using the SNP 9 primers, gDNA of 40 cases (20 controls, 9 BPDs and 11 MDDs) was sequenced with both the forward and reverse primers. The SNPs were located in exons a, b and c. SNPs b and c are intronic and found just before exon ‘b’ (7 bps upstream) and ‘c’ (12 bps upstream) respectively. Exon ‘a’ did not have a SNP in close proximity.
The genotypes were collected on an additional 26 bipolar genomic DNA samples extracted from lymphocytes from the National Institute of Mental Health (NIMH) for all 4 SNPs: SNP 6, SNP 9, SNP b and SNP c (see FIG. 26). A third cohort was genotyped consisting of the Stanley Foundation 105 dorsalateral prefrontal cortex (DLPFC) microarray samples (n=35 controls, n=35 bipolar disorder, n=35 schizophrenia) (Table 24). The Stanley samples were genotyped for SNP 9 and SNP b. For final analysis, the three groups of bipolar cases were combined and three control groups were merged and used for statistical comparisons of SNP 9 and SNP b. The results show an association of SNP 9 and SNP b haplotype with bipolar disorder and schizophrenia.
Genotype X Diagnosis Interaction for NCAM1 Splice Variants.
The splice variants are alternative splicing combinations of 3 mini-exons (a,b,c) with the fourth SEC exon are shown in FIG. 30. Two mood disorders were tested (Bipolar Disorder, Type I and Major Depressive Disorder, Recurrent) and both showed differences in NCAM1 splice variants in the DLPFC.
The present data relates NCAM1 polymorphic variation to bipolar disorder and splice variations in mRNA occurring near the polymorphisms. A genotypic association between SNP b in NCAM 1 and bipolar disorder and a suggestive association of SNP 9 with schizophrenia were found. Three of the two marker haplotypes for SNP 9 and SNP b, CT, C(T/C), and (C/A)(T/C) display varying frequency distribution between bipolar and controls. Schizophrenia and controls show differences in frequency distribution in four of the two marker haplotypes of SNP 9 and SNP b, CT, C(T/C), (C/A)(T/C) and (C/A)C. Bipolar disorder differs from schizophrenia for SNP 9 and SNP b by haplotype frequency differences. SNP b and SNP 9 are not in LD and they are individually related to schizophrenia (SNP 9) and bipolar disorder (SNP b).
The splice variant evidence for SNP 9 and b confirm that each SNP can be associated with differences in SEC exon splicing, thus providing some differential mechanisms for release of NCAM1 in the brain. We observed notable differences between polymorphisms in NCAM1 and the relative isoform variants of the SEC exon which can lead to truncation and secretion of NCAM1 in the brain. This finding concerning the difference in splice variant relative amounts as a function of certain genotypes was shown in three of the four SNPs where at least one genotype showed a difference in the amount of SEC by splice variant. This evidence suggests that the amount of SEC in brain is not regulated by just one genotype. Since the haplotypes composed of SNP 9 and SNP b are significantly different between controls and bipolar and between controls and schizophrenia this may support the observation of differential splicing patterns of the SEC exon found across many samples. Additionally, SNP 9 and SNP b are not in LD and thus the individual associations in schizophrenia and bipolar with these SNPs also may be transmitted through differential splicing patterns. The SEC exon was clearly regulated by certain combinations of mini-exons. We have identified discrete splice variants that can be further studied and are perhaps associated with regulatory intronic SNPs.
Example 8
Lithium has long been the drug of choice for treating manic-depressive illness (manic-depression; bipolar affective disorder, BPD). This Example shows non-human primate genes which exhibit differential expression in response to treatment with lithium. The results have implications for understanding the mood stabilizing effects of lithium in patients with manic depression.
Gene expression profiling was carried out on the anterior cingulate cortex (AnCg) using high-density oligonucleotide microarrays (Affymetrix GeneChips). We determined differential gene expression profiles of postmortem brains from lithium-treated healthy monkeys over those of untreated controls, and validated candidate genes against those known to be lithium-responsive or disease-selective.
Some of the candidate genes that responded to chronic lithium treatment were the same as those found with changed expression levels in postmortem brains of subjects with mood disorders. Our results show that the GSK3B signaling system is altered in BPD and that it is a physiological target of lithium. The observed GSK3B signaling system change thus constitutes an endophenotype that is likely to be common to BPD and schizophrenia, notwithstanding their clinical and phenotypic disparity. The results, by facilitating reconstruction of the genetic networks underlying BPD pathophysiology will facilitate the rational mood stabilizers targeting the signal transduction network via GSK3.
Major depressive disorder (MDD) and bipolar affective disorder (BPD) are two most severe mood disorders. MDD is characterized by clinical depression, while BPD is marked by recurrent and dramatic swings of emotional highs (mania) and lows (depression). For decades, lithium carbonate (Li2CO3, commonly known as “lithium”) has been the benchmark medication for mania (hyperactive, incoherent and delusional behavior). Lithium unlike other anti-manic treatment agents is unique in its ability to abort the manic condition and restore patient's balanced mental status. Although numerous hypotheses have been proposed accounting for the neuro-protective properties of lithium, the precise molecular mechanism(s) by which lithium elicits its “mood stabilizing” effects in manic-depressive patients remains obscure. We addressed the challenge using a microarray strategy because it can permit simultaneous detection of multiple lithium-responsive genes and pathways in drug-treated primates and direct comparison of the observed gene expression changes with those found in postmortem brains from subjects with BPD. Lithium carbonate suspension (Roxane Laboratories, Inc., Columbus, Ohio) diluted in fruit juice was administered orally (18 mg/kg body weight) to a targeted plasma level of 0.6 to 1.2 mgEq/mL. The animals received the drug twice a day for varying periods ranging from 4 months to 1 year and 5 months to circumvent their tendency to spit the drug out.
AnCg showed a total of 220 candidate transcripts (65 upregulated and 155 down-regulated). The candidate genes from AnCg are listed in Table 28. Ontological annotations mapped candidate genes to several different biological processes and pathways, including GSK3B signaling system, as predicted, in the AnCg.
AnCg involvement demonstrated in this study together with published reports of hippocampus involvement in lithium response implicates likely involvement of the limbic system in mood disorders as well as in the differential gene expression elicited by chronic lithium treatment. The present study also illuminates multiple interrelated functional networks of neuronal signaling pathways acting in unity with GSK3B as a pivotal functional switch regulating gene expression in behavioral diseases of apparent disparate phenotypic diversity, BPD and SZ. The results show that manipulating GSK3B could affect one or more of the inositol triphosphate, NF-kB family, mitochondrial apoptosis, and ubiquitin-proteasome pathways.
Example 9
V-Atpase Subunits as Gene Candidates of Interest for Major Depressive Disorder (MDD)
Of the 14 V-ATPase subunits that we have interrogated with Affy microarrays and Ulumina microarrays, 7 subunits (50%) are differentially expressed (P<0.05) in hippocampal MDD versus control on either the Affymetrix or Illumina arrays. Three of the 7 subunits are differentially expressed in MDD hippocampus on both the Affymetrix and Illumina arrays (see Table 29). Two of the V-ATPase subunits are also differentially expressed (P<0.05) in our Affy microarray study of monkey hippocampus (i.e., Table 30, showing chronic social stress versus no-stress comparison). These findings demonstrate that drugs now being developed to inhibit V-ATPase in patients with cancer and osteoporosis may also prove useful as novel antidepressants.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, Genbank sequences, patents, and patent applications cited herein are hereby incorporated by reference.







TABLE 1a







Subject Data for Cohort A.






















AnCg
DLPFC


subject



brain


3′/5′
3′/5′


ID
gender
age
diagnosis
pH
PMI
AnCg 18S/28S
ratio
ratio


















1881
M
69
BPD
6.91
11
0.68
1.36
1.21


2311
M
23
BPD
7.12
9
1.80
1.47
1.13


2466
M
26
BPD
6.92
19
1.35
1.75
1.64


2566
F
56
BPD
6.83
29
1.05
1.48
1.34


3038
M
52
BPD
7.05
28
1.04
1.37
1.20


3241
M
59
BPD
6.99
16
1.38
1.33
1.17














averages for BPDs
47.5

6.97
18.6
1.22
1.46
1.28















2861
F
60
Ct
6.99
24
1.25
2.07
1.80


3018
M
70
Ct
7.03
27
0.91
1.57
1.20


2169
M
18
Ct
6.97
22
1.61
1.62
1.18


2316
M
58
Ct
7.02
26
1.27
1.47
1.27


2292
M
55
Ct
6.89
15
1.45
1.29
1.18


2805
M
45
Ct
6.86
21
1.84
1.61
1.06


3196
M
44
Ct
6.87
23
1.26
1.67
1.22














averages for
50

6.95
22.6
1.37
1.61
1.27


controls






















2208
F
72
MD
7.13
21
1.26
1.83
1.19


2267
M
19
MD
7.11
18
2.05
1.75
1.44


2315
M
58
MD
6.93
24
1.34
1.52
1.31


3071
M
49
MD
7.00
31
1.58
1.44
1.25


3064
M
46
MD
6.91
27
2.18
1.28
1.19


3031
M
49
MD
7.19
27
1.60
1.60
1.04


2944
M
52
MD
6.82
16
1.37
1.91
1.43


3004
F
48
MD
6.95
37
1.31
1.81
1.20


3168
M
39
MD
6.79
28
1.34
1.49
1.19














averages for MDDs
48.0

6.98
25.4
1.56
1.63
1.25
















TABLE 1b







Subject Data for Cohort B.






















AnCg
DLPFC


subject
gen-


brain

AnCg
3′/5′
3′/5′


ID
der
age
diagnosis
pH
PMI
18S/28S
ratio
ratio
















3145
M
77
Ct
6.62
7



3281
F
70
Ct
6.9
21



3516
M
41
Ct
7.01
23



3519
M
65
Ct
6.88
14



3523
M
40
Ct
7.07
37



3572
M
49
Ct
6.68
28













averages
57

6.86
21.67



for controls


















3169
M
35
MD
7.04
25



3365
M
47
MD
7.25
29



3398
F
80
MD
6.68
15



3481
M
66
MD
7.05
32













averages
57

7.01
25.25



for MDDs
















TABLE 2







Microarray data for all FGF transcripts reliably detected in either


DLPFC or AnCg and summary data for confirmation studies.










DLPFC
AnCg












UniGene ID
Transcript
p-value
direction
p-value
direction















Hs.278954
FGF1
<0.01‡,†
Decreased
0.01
Decreased


Hs.284244
FGF2
NS

<0.01*,‡
Decreased


Hs.433252
FGF7
NS

NS



Hs.111
FGF9
<0.01
Increased
<0.01*
Increased


Hs.343809
FGF12
NS

<0.01*
Increased


Hs.6540
FGF13
NS

NS



Hs.223851
FGF14
0.05
Increased
NS



Hs.748
FGFR1
NS
Increased
NS



Hs.404081
FGFR2
<0.01*,‡,†
Decreased
<0.01*,‡
Decreased


Hs.1420
FGFR3
<0.01*,‡,†
Decreased
<0.01*
Decreased





*Met FDR multiple testing correction at the level of accepting 5% false positives.


‡Observation was confirmed in an independent cohort of MDD and control subjects given in Table 1b, meeting p-values of <0.05 in all cases indicated.


†Observation was confirmed by real-time PCR analysis with p < 0.05.


NS = not significant.













TABLE 3





BPD
























Gene Bank



UniGene ID
Gene Symbol
Gene Name
RefSeq ID
Acc. No.
LocusLink





Hs.407520
CHN2
chimerin (chimaerin) 2
NM_004067
U07223
1124


Hs.13351
LANCL1
LanC lantibiotic
NM_006055
Y11395
10314




synthetase component







C-like 1







(bacterial)





Hs.309090
SFRS7
splicing factor,
NM_006276
L41887
6432




arginine/serinerich







7, 35 kDa





Hs.7910
RYBP
RING1 and YY1
NM_012234
AL049940
23429




binding protein





Hs.150101
LAMP1
lysosomal-associated
NM_005561
J04182
3916




membrane







protein 1





Hs.90458
SPTLC1
serine palmitoyl
NM_006415
Y08685
10558




transferase, long







chain base subunit 1





Hs.406532
RPN2
ribophorin II
NM_002951
AL031659
6185


Hs.408883
SCN1B
sodium channel,
NM_001037
L10338
6324




voltage-gated, type I, beta





Hs.91971
CGEF2
cAMP-regulated
NM_007023
U78516
11069




guanine nucleotide







exchange factor







II





Hs.49117
—
hypothetical protein
—
AL080093
—




DKFZp564







N1662





Hs.84244
KCNB1
potassium volta
NM_004
L02840
3745






















DLPFC-
DLPFC-



UniGene ID
Chromosome
AnCg-BP
AnCg-MD
Criteria
BP
MD
Criteria





Hs.407520
Chr: 7p15.3
UP
NC
2a, 3





Hs.13351
Chr: 2q33–q35
UP
NC
1, 2a
UP
NC
2a


Hs.309090
Chr: 2p22.1



UP
NC
1, 2a, 3


Hs.7910
Chr: 3p14.2



UP
NC
1, 2a, 3


Hs.150101
Chr: 13q34
UP
NC
2a, 3





Hs.90458
Chr: 9q22.2



UP
NC
1, 2a, 3


Hs.406532
Chr: 20q12–q13.1
UP
NC
2a
UP
NC
1, 2a


Hs.408883
Chr: 19q13.1
DOWN
NC
1, 3





Hs.91971
Chr: 2q31–q32
DOWN
NC
1, 3





Hs.49117
—



UP
NC
1, 3


Hs.84244
Chr: 20q13.2
DOWN
NC
1, 3
















TABLE 4







MDD


























AnCg-
AnCg-

DLPFC-
DLPFC-



UniGene ID
Gene Symbol
Gene Name
RefSeq ID
Gene Bank Acc. No.
LocusLink
Chromosome
BP
MD
Criteria
BP
MD
Criteria






















Hs.5462
SLC4A4
solute carrier family 4, sodium
NM_003759
AF007216
8671
Chr: 4q21
NC
DOWN
2b
NC
DOWN
3




bicarbonate cotransporter,














member 4












Hs.44
PTN
pleiotrophin (heparin binding
NM_002825
M57399
5764
Chr: 7q33–q34



NC
DOWN
1, 2b




growth factor 8, neurite growth-














promoting factor 1)












Hs.144845
BBOX1
butyrobetaine (gamma), 2-
NM_003986
AF082868
8424
Chr: 11p14.2



NC
DOWN
1, 2b, 3




oxoglutarate dioxygenase














(gamma-butyrobetaine hydroxylase) 1












Hs.170133
FOXO1A
forkhead box O1A (rhabdomyosarcoma)
NM_002015
AF032885
2308
Chr: 13q14.1
NC
DOWN
1, 2b
NC
DOWN
1, 3


Hs.62192
F3
coagulation factor III (thromboplastin,
NM_001993
J02931
2152
Chr: 1p22–p21



NC
DOWN
1, 2b, 3




tissue factor)












Hs.166994
FAT
FAT tumor suppressor homolog
NM_005245
X87241
2195
Chr: 4q34–q35



NC
DOWN
1, 2b, 3




1 (Drosophila)












Hs.82002
EDNRB
endothelin receptor type B
NM_000115
S57283
1910
13q22
NC
DOWN
1, 2b
NC
DOWN
1, 2b, 3


Hs.403997
VIL2
villin 2 (ezrin)
NM_003379
X51521
7430
Chr: 6q25.2–q26



NC
DOWN
1, 2b


Hs.8022
TU3A
TU3A protein
NM_007177
AF035283
11170
Chr: 3p21.1
NC
DOWN
1, 2b





Hs.356876
GPR125
G protein-coupled receptor
XM_291111
AK027494
166647
4p15.32–p15.31



NC
DOWN
1, 2b, 3




125












Hs.450919
GPC5
glypican 5
NM_004466
U66033
2262
Chr: 13q32



NC
DOWN
1, 2b


Hs.414151
DAAM2
dishevelled associated activator
NM_015345
AB002379
23500
Chr: 6p21.1
NC
DOWN
2b
NC
DOWN
3




of morphogenesis 2












Hs.172089
PORIMIN
pro-oncosis receptor inducing
NM_052932
AL050161
114908
Chr: 11q22.1
NC
DOWN
1, 3







membrane injury gene












Hs.77546
ANKRD15
ankyrin repeat domain 15
NM_015158
D79994
23189
Chr: 9p24.3



NC
DOWN
1, 3


Hs.26208
COL16A1
collagen, type XVI, alpha 1
NM_001856
M92642
1307
Chr: 1p35–p34



NC
DOWN
1, 3


Hs.434494
SYNJ2
synaptojanin 2
NM_003898
AF039945
8871
Chr: 6q25.3
NC
DOWN
1, 3





Hs.434418
MYT1L
myelin transcription factor
—
AB029029
23040
Chr: 2p25.3
NC
UP
1, 3







1-like












Hs.78748
RIMS3
regulating synaptic membrane
—
D87074
9783
Chr: 1pter–p22.2
NC
UP
1, 3







exocytosis 3












Hs.436987
ZNF288
zinc finger protein 288
NM_015642
AL050276
26137
Chr: 3q13.2
NC
DOWN
1
NC
DOWN
1


Hs.391392
ID4
inhibitor of DNA binding 4,
NM_001546
AL022726
3400
Chr: 6p22–p21



NC
DOWN
1




dominant negative helix-loop-














helix protein












Hs.109052
C14orf2
chromosome 14 open reading
NM_004894
AF054175
9556
Chr: 14q32.33
NC
UP
1







frame 2












Hs.33455
PADI2
peptidyl arginine deiminase,
NM_007365
AB023211
11240
Chr: 1p35.2–p35.1
NC
DOWN
1







type II












Hs.438240
ZFYVE16
zinc finger, FYVE domain containing
NM_014733
AB002303
9765
Chr: 5p15.2–q14.3
NC
DOWN
1







16












Hs.75462
BTG2
BTG family, member 2
NM_006763
U72649
7832
Chr: 1q32



NC
DOWN
1
















TABLE 5





Growth Factor Pathway Genes



















UniGene ID
Gene Symbol
Gene Name
RefSeq Transcript ID
LocusLink





Hs.433326
IGFBP2
insulin-like growth factor
NM_000597
3485




binding protein 2,






36 kDa




Hs.16512
OGFRL1
opioid growth factor receptor-
NM_024576
79627




like 1




Hs.799
DTR
diphtheria toxin receptor
NM_001945
1839




(heparin-binding






epidermal growth factor-






like growth factor)




Hs.105689
LTBP2
latent transforming growth
NM_000428
4053




factor beta binding






protein 2




Hs.289019
LTBP3
latent transforming growth
NM_021070
4054




factor beta binding






protein 3




Hs.376032
PDGFA
platelet-derived growth
NM_002607///NM—
5154




factor alpha polypeptide
033023



Hs.839
IGFALS
insulin-like growth factor
NM_004970
3483




binding protein,






acid labile subunit




Hs.342874
TGFBR3
transforming growth
NM_003243
7049




factor, beta receptor






III (betaglycan, 300 kDa)




Hs.404081
FGFR2
fibroblast growth factor
NM_000141///NM—
2263




receptor 2 (bacteria-
022969///NM_022970///





expressed kinase,
NM_022971///





keratinocyte growth factor
NM_022972///





receptor, craniofacial
NM_022973///NM—





dysostosis 1,
022974///NM_022975///





Crouzon syndrome,
NM_022976///





Pfeiffer syndrome, Jackson-
NM_023028///





Weiss syndrome)
NM_023029///NM—






023030///NM_023031



Hs.433252
FGF7
fibroblast growth factor
NM_002009
2252




7 (keratinocyte growth






factor)




Hs.67896
OGFR
opioid growth factor receptor
NM_007346
11054


Hs.446350
TGFBRAP1
transforming growth
NM_004257
9392




factor, beta receptor






associated protein 1




Hs.194208
FRS3
fibroblast growth factor
NM_006653
10817




receptor substrate 3




Hs.169300
TGFB2
transforming growth
NM_003238
7042




factor, beta 2




Hs.411881
GRB14
growth factor receptor-
NM_004490
2888




bound protein 14




Hs.1420
FGFR3
fibroblast growth factor
NM_000142///NM—
2261




receptor 3 (achondroplasia,
022965





thanatophoric






dwarfism)




Hs.450230
IGFBP3
insulin-like growth factor
NM_000598
3486




binding protein 3




Hs.308053
IGF1
insulin-like growth factor
NM_000618
3479




1 (somatomedin






C)




Hs.419124
MET
met proto-oncogene
NM_000245
4233




(hepatocyte growth factor






receptor)




Hs.284244
FGF2
fibroblast growth factor
NM_002006
2247




2 (basic)




Hs.76473
IGF2R
insulin-like growth factor
NM_000876
3482




2 receptor




Hs.410037
CTGF
connective tissue growth
NM_001901
1490




factor




Hs.274313
IGFBP6
insulin-like growth factor
NM_002178
3489




binding protein 6




Hs.111
FGF9
fibroblast growth factor
NM_002010
2254




9 (glia-activating factor)




Hs.380833
IGFBP5
insulin-like growth factor
NM_000599
3488




binding protein 5




Hs.79095
EPS15
epidermal growth factor
NM_001981
2060




receptor pathway






substrate 15




Hs.2132
EPS8
epidermal growth factor
NM_004447
2059




receptor pathway






substrate 8




Hs.278954
FGF1
fibroblast growth factor
NM_000800///NM—
2246




1 (acidic)
013394///NM_033136///






NM_033137



Hs.343809
FGF12
fibroblast growth factor
NM_004113///NM—
2257




12
021032



Hs.7768
FIBP
fibroblast growth factor
NM_004214///NM—
9158




(acidic) intracellular
198897





binding protein




Hs.127842
HDGFRP3
hepatoma-derived growth
NM_016073
50810




factor, related protein 3




Hs.416959
HGS
hepatocyte growth factor-
NM_004712
9146




regulated tyrosine






kinase substrate




Hs.239176
IGF1R
insulin-like growth factor
NM_000875
3480




1 receptor




Hs.435795
IGFBP7
insulin-like growth factor
NM_001553
3490




binding protein 7




Hs.439109
NTRK2
neurotrophic tyrosine
NM_006180
4915




kinase, receptor, type 2




Hs.26776
NTRK3
neurotrophic tyrosine
NM_002530
4916




kinase, receptor, type 3




Hs.43080
PDGFC
platelet derived growth
NM_016205
56034




factor C




Hs.44
PTN
pleiotrophin (heparin
NM_002825
5764




binding growth factor






8, neurite growth-promoting






factor 1)///






pleiotrophin (heparin






binding growth factor






8, neurite growth-promoting






factor 1)




Hs.114360
TSC22
transforming growth
NM_006022///NM—
8848




factor beta-stimulated
183422





protein TSC-22




Hs.73793
VEGF
vascular endothelial
NM_003376
7422




growth factor



















DLPFC MD
DLPFC BP
AnCg MD
AnCg BP


UniGene ID
Chromosomal Location
% P
direction
direction
direction
direction





Hs.433326
Chr: 2q33–q34
25
up
none
up
down


Hs.16512
Chr: 6q13
28
down
none
none
down


Hs.799
Chr: 5q23
38
none
up
none
none


Hs.105689
Chr: 14q24
42
none
none
none
down


Hs.289019
Chr: 11q12
43
none
none
none
up


Hs.376032
Chr: 7p22
43
none
none
none
down


Hs.839
Chr: 16p13.3
48
none
none
none
down


Hs.342874
Chr: 1p33–p32
54
none
up
none
none


Hs.404081
Chr: 10q26
55
down
none
down
up


Hs.433252
Chr: 15q15–q21.1
59
none
down
none
none


Hs.67896
Chr: 20q13.3
59
none
down
none
down


Hs.446350
Chr: 2q12.2
61
up
up
none
none


Hs.194208
Chr: 6p21.1
64
none
down
up
down


Hs.169300
Chr: 1q41
68
none
none
down
none


Hs.411881
Chr: 2q22–q24
69
none
up
up
up


Hs.1420
Chr: 4p16.3
76
down
none
down
none


Hs.450230
Chr: 7p13–p12
76
none
down
none
down


Hs.308053
Chr: 12q22–q23
81
down
none
down
down


Hs.419124
Chr: 7q31
81
none
up
up
none


Hs.284244
Chr: 4q26–q27
84
none
up
down
none


Hs.76473
Chr: 6q26
92
none
none
down
down


Hs.410037
Chr: 6q23.1
93
down
none
none
up


Hs.274313
Chr: 12q13
94
none
none
up
down


Hs.111
Chr: 13q11–q12
99
up
up
up
none


Hs.380833
Chr: 2q33–q36
99
down
none
none
down


Hs.79095
Chr: 1p32
100
none
none
none
up


Hs.2132
Chr: 12q23–q24
100
down
up
down
none


Hs.278954
Chr: 5q31
100
down
none
down
none


Hs.343809
Chr: 3q28
100
none
none
none
none


Hs.7768
Chr: 11q13.1
100
none
none
none
up


Hs.127842
Chr: 15q11.2
100
none
none
up
none


Hs.416959
Chr: 17q25
100
none
none
none
up


Hs.239176
Chr: 15q25–q26
100
none
none
none
down


Hs.435795
Chr: 4q12
100
down
down
down
none


Hs.439109
Chr: 9q22.1
100
down
none
down
none


Hs.26776
Chr: 15q25
100
none
none
none
down


Hs.43080
Chr: 4q32
100
none
none
none
up


Hs.44
Chr: 7q33–q34///
100
down
none
down
none



Chr: 7q33–q34







Hs.114360
Chr: 13q14
100
none
none
up
none


Hs.73793
Chr: 6p12
100
down
none
down
none
















TABLE 6







GPCR Pathway Genes




















U95Av2 probe




Gene Bank Acc.




DLPFC-




set
UniGene ID
Gene Symbol
Gene Name
RefSeq ID
No.
LocusLink
Chromosome
AnCg-BP
AnCg-MD
BP
DLPFC-MD






















CINP
38604_at
Hs.1832
NPY
neuropeptide Y
NM_000905
NM_000905
4852
Chr: 7p15.1
UP





CINP
1430_at
Hs.12409
SST
somatostatin
NM_001048
NM_001048
6750
Chr: 3q28
UP





CINP
2083_at
Hs.203
CCKBR
cholecystokinin
NM_000731
BC000740
887
Chr: 11p15.4
UP









B receptor










CINP
39928_at
Hs.512145
GRM3
glutamate receptor,
NM_000840
NM_000840
2913
Chr: 7q21.1–q21.2
UP

UP







metabotropic 3










CINP
32623_at
Hs.167017
GABBR1
gamma-aminobutyric
NM_001470
NM_001470
2550
Chr: 6p21.31


UP







acid














(GABA) B receptor, 1










CINP
37095_r—
Hs.99855
FPRL1
formyl peptide
NM_001462
M88107
2358
Chr: 19q13.3–q13.4
DOWN






at


receptor-like 1










CINP
1198_at
Hs.82002
EDNRB
endothelin receptor
NM_000115
M74921
1910
Chr: 13q22



DOWN






type B










CINP
34297_at
Hs.406094
GPR37
G protein-coupled
NM_005302
T16257
2861
Chr: 7q31
UP
DOWN
UP
DOWN






receptor














37 (endothelin














receptor type














B-like)










CINP
40240_at
Hs.448805
GPRC5B
G protein-coupled
NM_016235
AF202640
51704
Chr: 16p12
UP
DOWN
UP
DOWN






receptor,














family C, group














5, member B










CINP
38580_at
Hs.469951
GNAQ
guanine nucleotide
NM_002072
U40038
2776
Chr: 9q21
UP


UP






binding














protein (G protein),














q polypeptide










CINP
38279_at
Hs.437081
GNAZ
guanine nucleotide
NM_002073
NM_002073
2781
Chr: 22q11.22
UP









binding














protein (G protein),














alpha z














polypeptide










CINP
38176_at
Hs.155090
GNB5
guanine nucleotide
NM_006578
NM_016194
10681
Chr: 15q21.1


UP
UP






binding














protein (G protein),














beta 5











41086_at
Hs.141492
RGS20
regulator of G-
NM_003702
AF074979
8601
Chr: 8q12.1

DOWN

DOWN






protein signalling














20











34272_at
Hs.386726
RGS4
regulator of G-
NM_005613
AL514445
5999
Chr: 1q23.2



UP






protein signalling 4










CINP
36311_at
Hs.416061
PDE1A
phosphodiesterase
NM_005019
NM_005019
5136
Chr: 4
UP









1A, calmodulin-














dependent











32645_at
Hs.502577
PDE4DIP
phosphodiesterase
XM_380170
NM_022359
9659
Chr: 1q12

UP








4D interacting














protein














(myomegalin)











37676_at
Hs.78746
PDE8A
phosphodiesterase
NM_002605
BE568219
5151
Chr: 15q25.2



DOWN






8A










CINP
226_at
Hs.280342
PRKAR1A
protein kinase,
NM_002734
AL050038
5573
Chr: 17q23–q24


UP







cAMP-dependent,














regulatory,














type I, alpha














(tissue specific














extinguisher














1)










CINP
1383_at
Hs.512628
PPP2R2A
protein phosphatase
NM_002717
NM_002717
5520
Chr: 8p21.1
UP

UP







2 (formerly














2A), regulatory














subunit














B (PR 52), alpha














isoform











39364_s—
Hs.303090
PPP1R3C
protein phosphatase
NM_005398
N26005
5507
Chr: 10q23–q24

DOWN

DOWN



at


1, regulatory














(inhibitor)














subunit 3C











39780_at
Hs.187543
PPP3CB
protein phosphatase
NM_021132
NM_021132
5532
Chr: 10q21–q22

UP








3 (formerly














2B), catalytic














subunit,














beta isoform














(calcineurin A














beta)










CINP
40704_at
Hs.85701
PIK3CA
phosphoinositide-
NM_006218
NM_006218
5290
Chr: 3q26.3
DOWN









3-kinase,














catalytic, alpha














polypeptide










CINP
33628_g—
Hs.426967
PIK3CD
phosphoinositide-
NM_005026
U86453
5293
Chr: 1p36.2
DOWN






at


3-kinase,














catalytic, delta














polypeptide










CINP
146_at
Hs.154846
PIK4CB
phosphatidylinositol
NM_002651
NM_002651
5298
Chr: 1q21
UP









4-kinase,














catalytic, beta














polypeptide










CINP
40217_s—
Hs.380684
CDS1
CDP-diacylglycerol
NM_001263
NM_001263
1040
Chr: 4q21.23
DOWN






at


synthase














(phosphatidate














cytidylyltransferase) 1










CINP
656_at
Hs.32309
INPP1
inositol polyphosphate-
NM_002194
NM_002194
3628
Chr: 2q32
UP









1-phosphatase










CINP
118_at
Hs.2722
ITPKA
inositol 1,4,5-trisphosphate
NM_002220
NM_002220
3706
Chr: 15q14–q21



UP






3-kinase A











33508_at
Hs.334575
INPP4A
inositol polyphosphate-
NM_001566
AA355179
3631
Chr: 2q11.2



UP






4-phosphatase,














type














I, 107 kDa











32779_s—
Hs.149900
ITPR1
inositol 1,4,5-triphosphate
NM_002222
NM_002222
3708
Chr: 3p26–p25

UP





at


receptor,














type 1











353_at
Hs.7370
PITPNB
phosphotidylinositol
NM_012399
AL031591
23760
Chr: 22q12.1



UP






transfer














protein, beta











1217_g_at
Hs.349845
PRKCB1
protein kinase
NM_002738
NM_002738
5579
Chr: 16p11.2

UP

UP






C, beta 1











362_at
Hs.407181
PRKCZ
protein kinase
NM_002744
NM_002744
5590
Chr: 1p36.33–p36.2

UP








C, zeta










CINP
976_s_at
Hs.324473
MAPK1
mitogen-activated
NM_002745
NM_002745
5594
Chr: 22q11.2
UP


UP






protein kinase 1










CINP
1844_s_at
Hs.132311
MAP2K1
mitogen-activated
NM_002755
AI571419
5604
Chr: 15q22.1–q22.33
UP


UP






protein kinase














kinase 1










CINP
1712_s_at
Hs.134106
MAP2K4
mitogen-activated
NM_003010
NM_003010
6416
Chr: 17p11.2
UP


UP






protein kinase














kinase 4










CINP
1557_at
Hs.64056
PAK1
p21/Cdc42/R
NM_002576
U51120
5058
Chr: 11q13–q14
UP
UP

UP






ac1-activated














kinase 1 (STE














20 homolog, y














east)










CINP
33073_at
Hs.152663
PAK3
p21 (CDKN1A)-
NM_002578
AW085556
5063
Chr: Xq22.3–q23
UP
UP
UP
UP






activated kinase 3










CINP
39736_at
Hs.355832
CDC42
cell division cycle
NM_001791
NM_015858
998
Chr: 1p36.1

UP
UP







42 (GTP














binding protein,














25 kDa)










CINP
1590_s_at
Hs.37003
HRAS
v-Ha-ras Harvey
NM_005343
NM_005343
3265
Chr: 11p15.5
UP
UP








rat sarcoma














viral oncogene














homolog
















TABLE 7







GROWTH FACTOR



















Gene
MDD




BPD






UniGene ID
Symbol
Amy
AnCg
DLPFC
HC
nAcc
Amy
AnCg
DLPFC
HC
nAcc










Epidermal Growth Factor System


















Hs.419815
EGF



−1.1








Hs.79095
EPS15









1.3


Hs.147176
EPS15R












Hs.2132
EPS8

−1.2










Hs.799
DTR





−1.1











Fibroblast Growth Factor System


















Hs.278954
FGF1
−1.2


−1.4








Hs.343809
FGF12
1.5

1.5









Hs.6540
FGF13




1.5



1.5



Hs.223851
FGF14
1.5




1.3


1.4



Hs.284244
FGF2
−1.4
−1.3

−1.4





−1.8


Hs.433252
FGF7







−1.1




Hs.111
FGF9
1.4
1.2
1.3
1.6








Hs.748
FGFR1












Hs.404081
FGFR2
−1.1
−1.3
−1.2
−1.3








Hs.1420
FGFR3
−1.2
−1.4
−1.5









Hs.7768
FIBP



1.3








Hs.194208
FRS3

















Insulin-Like Growth Factor System


















Hs.308053
IGF1












Hs.239176
IGF1R



−1.2

−1.3






Hs.76473
IGF2R












Hs.839
IGFALS












Hs.433326
IGFBP2

1.4










Hs.450230
IGFBP3
−1.3




−1.4






Hs.1516
IGFBP4





−1.6






Hs.380833
IGFBP5

−1.2
−1.2









Hs.274313
IGFBP6












Hs.435795
IGFBP7

















Neurotrophins


















Hs.439027
BDNF












Hs.439109
NTRK2
−1.2
−1.4
−1.3
−1.3








Hs.26776
NTRK3












Hs.194774
CNTFR

















Opioid Growth Factor System


















Hs.67896
OGFR












Hs.16512
OGFRL1


−1.2



−1.2










Platelet-Derived Growth Factor System


















Hs.1976
PDGFB












Hs.43080
PDGFC












Hs.74615
PDGFRA
−1.3


−1.3


−1.2

−1.3



Hs.307783
PDGFRB

















Transforming Growth Factor System


















Hs.25195
EBAF












Hs.435067
ECGF1












Hs.170009
TGFA








−1.1



Hs.169300
TGFB2












Hs.421496
TGFBI





−1.9






Hs.82028
TGFBR2












Hs.342874
TGFBR3





−1.3






Hs.446350
TGFBR













AP1












Hs.114360
TSC22












Hs.241257
LTBP1





−1.2
−1.1





Hs.105689
LTBP2





−1.2






Hs.289019
LTBP3

















Vascular Endothelial Growth Factor System


















Hs.73793
VEGF



−1.4








Hs.78781
VEGFB












Hs.79141
VEGFC

















Other Growth Factors/Receptors


















Hs.89525
HDGF












Hs.127842
HDGFR



1.3
1.3



1.3
1.2



P3












Hs.44
PTN

−1.2










Hs.270833
AREG












Hs.410037
CTGF

















Other Growth Factor Receptor Signalling Proteins















Hs.512118
GRB10









Hs.411881
GRB14









Hs.411366
GRB2
1.1








Hs.416959
HGS






1.1


Hs.419124
MET
















TABLE 8







GLU/GABA


GABA/glutamate signaling genes in Mood Disorders - SFN genes For IDF inclusion









UniGene
BPD
MDD




















ID

Gene

Common name
Locus
AnCg
DLPFC
HC
Amy
AnCg
DLPFC
HC
Amy






GABAergic:















GABA A receptor














Hs.24969
alpha 5
GABRA5
GABA (A)
GABA-A-Ra5
15q11.2
1.58
1.64



1.58







receptor,















alpha 5












Hs.302352
beta 3
GABARB3
GABA (A)
GABA-A-Rb3
15q11.2





1.54







receptor,















beta 3












Hs.7195
gamma 2
GABARG2
GABA (A)
GABA-A-Rg2
5q3.1




1.58
1.64

1.59





receptor,















gamma 2













Glutamate















receptor














Hs.7117
ionotropic,
GRIA1

IGluR1
5q31.1





1.18





AMPA1














Hs.512145
Glutamate
GRM3
Glutamate
mGluR3
7q21.1

1.22

1.32







receptor,

receptor,













metabotropic 3

metabotropic 3













Glutamate















transporters














Hs.349088
Glutamate
SLC1A2
Solute carrier
GLT-1;
11p13


0.84

0.71

0.87
0.81



transporter,

family 1 (glial
EAAT2












Na+-dependent,

high affinity













EAAT2














Hs.371369
Glutamate
SLC1A3
Solute carrier
GLAST;
5p13




0.65






Transporter,

family 1 (glial
EAAT1












Na+-dependent,

high affinity -













EAAT1















Glutamine















synthetase














Hs.442669
Glutamate-
GLUL
Glutamate-
Glutamine
1q31




0.72


0.86



ammonia

ammonia
synthetase












ligase

ligase (glut















synthetase)





GABA, gamma amino butyric acid; glut/glutamate, glutamic acid; MDD, major depressive disorder; BPD, bipolar disorder


AnCg, anterior cingulate cortex; DLPFC, dorso-lateral prefrontal cortex; HC, hippocampus; Amy, amygdala.


Numbers (Fold Change) in RED denote INCREASES, and BLUE the DECREASES













TABLE 9





Pathway Analysis






















Gene Category
System
Rank
EASE score
Gene Category
System
Rank
EASE score





Dorsolareral Prefrontal Cortex









Bipolar Disorder



Major Depressive Disorder





PKC

1st
0.00495
Hs_Glutamate Metabolism
GenMAPP
1st
0.338


Hs_G Protein Signaling
GenMAPP
2nd
0.121
Hs_G13 Signaling Pathway
GenMAPP
2nd
0.387


Hs_Electron Transport Chain
GenMAPP
3rd
0.212
G protein beta, gamma

3rd
0.478


Hs_Ribosomal Proteins
GenMAPP
4th
0.29
Hs_Peptide GPCRs
GenMAPP
4th
0.566


Hs_Wnt signaling
GenMAPP
5th
0.362
Hs_G Protein Signaling
GenMAPP
5th
0.571


Anterior Cingulate Cortex









Bipolar Disorder



Major Depressive Disorder





Hs_Electron Transport Chain
GenMAPP
1st
0.0972
GPCR-orphan

1st
0.0407


Hs_Ribosomal Proteins
GenMAPP
2nd
0.102
Hs_Orphan GPCRs
GenMAPP
2nd
0.0447


GPCR-orphan

3rd
0.289
Hs_Electron Transport Chain
GenMAPP
3rd
0.0677


PKA

4th
0.368
GPCR

4th
0.189


Hs_Proteasome Degradation
GenMAPP
5th
0.427
PI signaling

5th
0.198


Amygdala









Bipolar Disorder



Major Depressive Disorder





Hs_Nuclear Receptors
GenMAPP
1st
0.0826
Hs_Glycolysis and Gluconeogenesis
GenMAPP
1st
0.0022


Hs_Apoptosis
GenMAPP
2nd
0.228
Hs_Krebs-TCA Cycle
GenMAPP
2nd
0.00447


GPCR-orphan

3rd
0.46
Hs_Electron Transport Chain
GenMAPP
3rd
0.0563


GPCR

4th
0.555
PI signaling

4th
0.0846


Hs_Krebs-TCA Cycle
GenMAPP
5th
0.61
Hs_G Protein Signaling
GenMAPP
5th
0.154


Hippocampus









Bipolar Disorder



Major Depressive Disorder





Hs_Electron Transport Chain
GenMAPP
1st
0.00000276
Hs_Electron Transport Chain
GenMAPP
1st
2.41E−07


Hs_Glycolysis and
GenMAPP
2nd
0.00838
Hs_Glycolysis and Gluconeogenesis
GenMAPP
2nd
0.0059


Gluconeogenesis









cAMP signaling

3rd
0.134
Rn_Ribosomal Proteins
GenMAPP
3rd
0.16


PKA

4th
0.14
Hs_Krebs-TCA Cycle
GenMAPP
4th
0.179


GPCR

5th
0.187
MAPK signaling

5th
0.22










Arrray Data
















Large

p-value(BP -
FC(BP -



NAME
SYMBOL
Category
Subcategory
Control)
Control)





Dorsolateral Prefrontal Cortex








Bipolar Disorder vs Control








UniGene Cluster








Hs.458426
cholecystokinin
CCK
Ligand
Ligand-neuropeptide
0.021
1.164


Hs.155090
guanine nucleotide binding protein (G
GNB5
G protein
GNB
0.025
1.210



protein), beta 5

beta, gamma





Hs.434387
protein kinase C, nu
PRKCN
PI signaling
PKC
0.023
1.081


Hs.496511
protein kinase C, iota
PRKCI
PI signaling
PKC
0.048
1.184


Hs.408049
protein kinase C, theta
PRKCQ
PI signaling
PKC
0.046
1.104


Hs.2890
protein kinase C, gamma
PRKCG
PI signaling
PKC
0.032
0.757


Major Depressive Disorder vs








Control








UniGene Cluster








Hs.458426
cholecystokinin
CCK
Ligand
Ligand-neuropeptide
0.015
1.152


Hs.82002
endothelin receptor type B
EDNRB
GPCR
GPCR-neuropeptide
0.000
0.550


Hs.184841
somatostatin receptor 2
SSTR2
GPCR
GPCR-neuropeptide
0.007
1.142


Hs.448805
G protein-coupled receptor, family C,
GPRC5B
GPCR
GPCR-orphan
0.009
0.694



group 5, member B







Hs.6527
G protein-coupled receptor 56
GPR56
GPCR
GPCR-orphan
0.010
0.790


Hs.155090
guanine nucleotide binding protein (G
GNB5
G protein
GNB
0.024
1.184



protein), beta 5

beta, gamma





Hs.8107
guanine nucleotide binding protein (G
GNG12
G protein
GNG
0.004
0.743



protein), gamma 12

beta, gamma





Hs.141492
regulator of G-protein signalling 20
RGS20
G protein
RGS
0.007
0.582





regulator





Anterior Cingulate Cortex








Bipolar Disorder vs Control








UniGene Cluster








Hs.203
cholecystokinin B receptor
CCKBR
GPCR
GPCR-neuropeptide
0.019
1.215


Hs.188
phosphodiesterase 4B, cAMP-specific
PDE4B
cAMP
phosphodiesterase
0.026
0.880



(phosphodiesterase E4 dunce homolog,

signaling






Drosophila)







Hs.350631
A kinase (PRKA) anchor protein 13
AKAP13
cAMP
PKA
0.019
0.887





signaling





Hs.440404
protein kinase, cAMP-dependent,
PRKAR2A
cAMP
PKA
0.007
0.954



regulatory, type II, alpha

signaling





Hs.183994
protein phosphatase 1, catalytic subunit,
PPP1CA
cAMP
PP related
0.004
1.152



alpha isoform

signaling





Hs.435238
protein phosphatase 1, regulatory
PPP1R1A
cAMP
PP related
0.017
1.233



(inhibitor) subunit 1A

signaling





Major Depressive Disorder vs








Control








UniGene Cluster








Hs.82002
endothelin receptor type B
EDNRB
GPCR
GPCR-neuropeptide
0.001
0.565


Hs.203
cholecystokinin B receptor
CCKBR
GPCR
GPCR-neuropeptide
0.011
1.205


Hs.406094
G protein-coupled receptor 37
GPR37
GPCR
GPCR-orphan
0.003
0.595


Hs.448805
G protein-coupled receptor, family C,
GPRC5B
GPCR
GPCR-orphan
0.000
0.662



group 5, member B







Hs.6527
G protein-coupled receptor 56
GPR56
GPCR
GPCR-orphan
0.002
0.787


Hs.46332
G protein-coupled receptor 6
GPR6
GPCR
GPCR-orphan
0.042
1.249


Hs.160271
G protein-coupled receptor 48
GPR48
GPCR
GPCR-orphan
0.000
0.713


Hs.166705
G protein-coupled receptor 49
GPR49
GPCR
GPCR-orphan
0.011
0.839


Hs.198612
G protein-coupled receptor 51
GPR51
GPCR
GPCR-orphan
0.003
1.173


Hs.155090
guanine nucleotide binding protein (G
GNB5
G protein
GNB
0.010
1.199



protein), beta 5

beta, gamma





Hs.149900
inositol 1,4,5-triphosphate receptor, type 1
ITPR1
PI signaling
IP3 receptor
0.003
1.227


Hs.153687
inositol polyphosphate-4-phosphatase,
INPP4B
PI signaling
Phosphatidylinositol
0.001
1.246



type II, 105 kDa


metabolism




Hs.151408
phospholipase C, beta 4
PLCB4
PI signaling
Phosphatidylinositol
0.005
1.215






metabolism




Hs.52463
inositol polyphosphate-5-phosphatase F
INPP5F
PI signaling
Phosphatidylinositol
0.003
1.181






metabolism




Hs.158318
diacylglycerol kinase, beta 90 kDa
DGKB
PI signaling
Phosphatidylinositol
0.024
1.192






metabolism




Hs.249235
phosphoinositide-3-kinase, class 2,
PIK3C2A
PI signaling
Phosphatidylinositol
0.027
0.770



alpha polypeptide


metabolism




Hs.408063
inositol polyphosphate-5-phosphatase,
INPP5A
PI signaling
Phosphatidylinositol
0.011
1.126



40 kDa


metabolism




Hs.239818
phosphoinositide-3-kinase, catalytic,
PIK3CB
PI signaling
Phosphatidylinositol
0.021
1.084



beta polypeptide


metabolism




Hs.349845
protein kinase C, beta 1
PRKCB1
PI signaling
PKC
0.014
1.125


Hs.79000
growth associated protein 43
GAP43
PI signaling
PKC related
0.006
1.133


Amygdala








Bipolar Disorder vs Control








Column ID








Hs.12409
somatostatin
SST
Ligand
Ligand-neuropeptide
0.011
1.672


Hs.46453
G protein-coupled receptor 17
GPR17
GPCR
GPCR-orphan
0.034
1.457


Hs.118552
likely ortholog of rat GRP78-binding
GBP
GPCR
GPCR-orphan
0.009
1.170



protein







Major Depressive Disorder vs








Control








UniGene Cluster








Hs.22584
prodynorphin
PDYN
Ligand
Ligand-neuropeptide
0.003
2.184


Hs.131138
neurotensin receptor 2
NTSR2
GPCR
GPCR-neuropeptide
0.001
0.684


Hs.82002
endothelin receptor type B
EDNRB
GPCR
GPCR-neuropeptide
0.005
0.706


Hs.46453
G protein-coupled receptor 17
GPR17
GPCR
GPCR-orphan
0.003
0.661


Hs.6527
G protein-coupled receptor 56
GPR56
GPCR
GPCR-orphan
0.000
0.781


Hs.448805
G protein-coupled receptor, family C,
GPRC5B
GPCR
GPCR-orphan
0.039
0.870



group 5, member B







Hs.155090
guanine nucleotide binding protein (G
GNB5
G protein
GNB
0.000
1.295



protein), beta 5

beta, gamma





Hs.8107
guanine nucleotide binding protein (G
GNG12
G protein
GNG
0.001
0.732



protein), gamma 12

beta, gamma





Hs.50612
guanine nucleotide binding protein (G
GNA14
PI signaling
GNA
0.007
0.691



protein), alpha 14







Hs.149900
inositol 1,4,5-triphosphate receptor, type 1
ITPR1
PI signaling
IP3 receptor
0.003
1.454


Hs.158318
diacylglycerol kinase, beta 90 kDa
DGKB
PI signaling
Phosphatidylinositol
0.001
1.444






metabolism




Hs.78877
inositol 1,4,5-trisphosphate 3-kinase B
ITPKB
PI signaling
Phosphatidylinositol
0.001
0.727






metabolism




Hs.2722
inositol 1,4,5-trisphosphate 3-kinase A
ITPKA
PI signaling
Phosphatidylinositol
0.011
1.461






metabolism




Hs.408063
inositol polyphosphate-5-phosphatase,
INPP5A
PI signaling
Phosphatidylinositol
0.003
1.358



40 kDa


metabolism




Hs.429643
phospholipase C, beta 1
PLCB1
PI signaling
Phosphatidylinositol
0.004
1.203



(phosphoinositide-specific)


metabolism




Hs.249235
phosphoinositide-3-kinase, class 2,
PIK3C2A
PI signaling
Phosphatidylinositol
0.013
0.782



alpha polypeptide


metabolism




Hs.52463
inositol polyphosphate-5-phosphatase F
INPP5F
PI signaling
Phosphatidylinositol
0.004
1.282






metabolism




Hs.334575
inositol polyphosphate-4-phosphatase,
INPP4A
PI signaling
Phosphatidylinositol
0.013
1.138



type I, 107 kDa


metabolism




Hs.25156
inositol polyphosphate-5-phosphatase,
INPP5E
PI signaling
Phosphatidylinositol
0.014
0.914



72 kDa


metabolism




Hs.349845
protein kinase C, beta 1
PRKCB1
PI signaling
PKC
0.002
1.600


Hs.512640
protein kinase C substrate 80K-H
PRKCSH
PI signaling
PKC
0.004
0.916


Hs.349611
protein kinase C, alpha
PRKCA
PI signaling
PKC
0.005
0.923


Hippocampus








Bipolar Disorder vs Control








UniGene Cluster








Hs.296341
CAP, adenylate cyclase-associated
CAP2
cAMP
adenylate cyclase
0.035
1.396



protein, 2 (yeast)

signaling
related




Hs.13313
cAMP responsive element binding
CREBL2
cAMP
CRE
0.033
1.341



protein-like 2

signaling





Hs.203862
guanine nucleotide binding protein (G
GNAI1
cAMP
GNA
0.000
2.075



protein), alpha inhibiting activity

signaling






polypeptide 1







Hs.157307
GNAS complex locus
GNAS
cAMP
GNA
0.038
1.127





signaling





Hs.416467
phosphodiesterase 10A
PDE10A
cAMP
phosphodiesterase
0.001
0.921





signaling





Hs.386791
phosphodiesterase 3A, cGMP-inhibited
PDE3A
cAMP
phosphodiesterase
0.005
0.790





signaling





Hs.337616
phosphodiesterase 3B, cGMP-inhibited
PDE3B
cAMP
phosphodiesterase
0.009
0.835





signaling





Hs.43322
protein kinase, AMP-activated, alpha 1
PRKAA1
cAMP
PKA
0.001
0.880



catalytic subunit

signaling





Hs.350631
A kinase (PRKA) anchor protein 13
AKAP13
cAMP
PKA
0.001
0.806





signaling





Hs.280342
protein kinase, cAMP-dependent,
PRKAR1A
cAMP
PKA
0.003
1.569



regulatory, type I, alpha (tissue specific

signaling






extinguisher 1)







Hs.156324
protein kinase, cAMP-dependent,
PRKACB
cAMP
PKA
0.012
1.418



catalytic, beta

signaling





Hs.3136
protein kinase, AMP-activated, gamma
PRKAG1
cAMP
PKA
0.014
1.225



1 non-catalytic subunit

signaling





Hs.433700
protein kinase (cAMP-dependent,
PKIA
cAMP
PKA related
0.014
1.385



catalytic) inhibitor alpha

signaling





Hs.79081
protein phosphatase 1, catalytic subunit,
PPP1CC
cAMP
PP related
0.001
1.426



gamma isoform

signaling





Hs.267819
protein phosphatase 1, regulatory
PPP1R2
cAMP
PP related
0.002
1.933



(inhibitor) subunit 2

signaling





Hs.187543
protein phosphatase 3 (formerly 2B),
PPP3CB
cAMP
PP related
0.007
1.476



catalytic subunit, beta isoform

signaling






(calcineurin A beta)







Hs.166071
cyclin-dependent kinase 5
CDK5
cAMP
PP related
0.017
1.296





signaling





Hs.21537
protein phosphatase 1, catalytic subunit,
PPP1CB
cAMP
PP related
0.005
1.334



beta isoform

signaling





Hs.272458
protein phosphatase 3 (formerly 2B),
PPP3CA
cAMP
PP related
0.017
1.357



catalytic subunit, alpha isoform

signaling






(calcineurin A alpha)







Hs.431156
protein phosphatase 2 (formerly 2A),
PPP2R1B
cAMP
PP related
0.017
0.927



regulatory subunit A (PR 65), beta

signaling






isoform







Hs.380764
protein phosphatase 2 (formerly 2A),
PPP2R2B
cAMP
PP related
0.009
1.215



regulatory subunit B (PR 52), beta

signaling






isoform







Hs.118244
protein phosphatase 2, regulatory
PPP2R5D
cAMP
PP related
0.008
1.118



subunit B (B56), delta isoform

signaling





Hs.356590
protein phosphatase 1, regulatory
PPP1R8
cAMP
PP related
0.011
1.210



(inhibitor) subunit 8

signaling





Hs.76556
protein phosphatase 1, regulatory
PPP1R15A
cAMP
PP related
0.045
0.828



(inhibitor) subunit 15A

signaling





Major Depressive Disorder vs








Control








UniGene Cluster








Hs.234521
mitogen-activated protein kinase-
MAPKAPK3

MAPK
0.007
0.845



activated protein kinase 3







Hs.324473
mitogen-activated protein kinase 1
MAPK1
MAPK
MAPK
0.007
1.612





signaling





Hs.348446
mitogen-activated protein kinase 9
MAPK9

MAPK
0.002
1.630


Hs.75074
mitogen-activated protein kinase-
MAPKAPK2

MAPK
0.006
0.872



activated protein kinase 2







Hs.271980
mitogen-activated protein kinase 6
MAPK6

MAPK
0.004
1.471


Hs.25209
mitogen-activated protein kinase 10
MAPK10
MAPK
MAPK
0.012
1.374





signaling





Hs.134106
mitogen-activated protein kinase kinase 4
MAP2K4
MAPK
MAPKK
0.004
1.657





signaling





Hs.432453
mitogen-activated protein kinase kinase
MAP3K8

MAPKKK
0.004
0.807



kinase 8







Hs.437214
mitogen-activated protein kinase kinase
MAP3K9

MAPKKK
0.008
1.162



kinase 9







Hs.28827
mitogen-activated protein kinase kinase
MAP3K2

MAPKKK
0.004
0.873



kinase 2







Hs.403927
mitogen-activated protein kinase kinase
MAP3K7IP1

MAPKKK
0.041
0.847



kinase 7 interacting protein 1







Hs.206097
related RAS viral (r-ras) oncogene
RRAS2
MAPK
RAS-MAPK
0.002
0.863



homolog 2

signaling





Hs.37003
v-Ha-ras Harvey rat sarcoma viral
HRAS
MAPK
RAS-MAPK
0.008
1.196



oncogene homolog

signaling





Hs.412107
v-Ki-ras2 Kirsten rat sarcoma 2 viral
KRAS2
MAPK
RAS-MAPK
0.013
1.451



oncogene homolog

signaling










qRT-PCR









Fold Change


















AnCg

DLPFC




Symbol
UniGene
Gene Name
U133A
qRT-PCR
U133A
qRT-PCR






Bipolar Disorder vs









Control









CCKBR
Hs.203
Cholecystokinin B receptor
1.21{circumflex over ( )}
1.44**





Major Depressive









Disorder vs









Control









EDNRB
Hs.82002
Endothelin receptor type B
0.57
0.63*
0.55{circumflex over ( )}
0.76



CCKBR
Hs.203
cholecystokinin B receptor
1.21
1.09





GPR37
Hs.406094
G protein-coupled receptor 37
0.59{circumflex over ( )}
0.74





GPRC5B
Hs.448805
G protein-coupled receptor C-5-B
0.66{circumflex over ( )}
0.65**
0.69{circumflex over ( )}
0.85



GNB5
Hs.155090
G protein, beta 5
1.20
1.09
1.18{circumflex over ( )}
1.61**



RGS20
Hs.141492
Regulator of G-protein signalling 20


0.58{circumflex over ( )}
0.70*





{circumflex over ( )}Microarray U133A, ANOVA p < 0.05


*qRT-PCR, one tailed Student's t-test, p < 0.1


**qRT-PCR, one tailed Student's t-test, p < 0.05






















TABLE 10





Duplicate 1


Differential Expression








1 = No
Mood And Region
Hs. Unigene
Value
Rank
Accession
UniGene_ID
LocusLink_ID
Symbol
Description



























Probe_ID (BPDv C) AnCg
Hs.20021_at
−7.727057737
9
R17168
Hs.20021
6843
VAMP1
VESICLE-ASSOCIATED MEMBRANE












PROTEIN 1












(SYNAPTOBREVIN 1)




Probe_ID (BPDv
Hs.119316_at
7.019916677
17
AI075114
Hs.119316
5188
PET112L
PET112-LIKE (YEAST)




C) AnCg




Probe_ID (BPDv
Hs.194673_at
6.739939746
24
AA954646
Hs.194673
8682
PEA15
PHOSPHOPROTEIN ENRICHED IN




C) AnCg







ASTROCYTES 15




Probe_ID (BPDv C) AnCg
Hs.356231_at
−6.670345597
28
AI038963
Hs.356231
79085
SLC25A23
SOLUTE CARRIER FAMILY 25












(MITOCHONDRIAL CARRIER;












PHOSPHATE CARRIER), MEMBER












23




Probe_ID (BPDv
Hs.438303_at
5.313202083
72
AA969386
Hs.438303
23395
LARS2
LEUCYL-TRNA SYNTHETASE 2,




C) AnCg







MITOCHONDRIAL




Probe_ID (BPDv
Hs.109052_at
5.019252834
103
AA865834
Hs.109052
9556
C14orf2
CHROMOSOME 14 OPEN




C) AnCg







READING FRAME 2




Probe_ID (BPDv
Hs.76366_at
4.693419228
127
AA999694
Hs.76366
572
BAD
BCL2-ANTAGONIST OF CELL




C) AnCg







DEATH




Probe_ID (BPDv
Hs.279939_at
4.582420323
138
AA977776
Hs.279939
23787
MTCH1
MITOCHONDRIAL CARRIER




C) AnCg







HOMOLOG 1 (C. ELEGANS)




Probe_ID (BPDv
Hs.74047_at
4.496465885
150

Hs.74047




C) AnCg




Probe_ID (BPDv C) AnCg
Hs.3100_at
4.309487236
169
AA954213
Hs.3100
3735
KARS
LYSYL-TRNA SYNTHETASE




Probe_ID (BPDv
Hs.243491_at
−4.224934266
183

Hs.243491




C) AnCg




Probe_ID (BPDv
Hs.3254_at
4.193592918
195
AA971301
Hs.3254
6150
MRPL23
MITOCHONDRIAL RIBOSOMAL




C) AnCg







PROTEIN L23




Probe_ID (BPDv
Hs.177584_at
4.165654802
200
AA961910
Hs.177584
5019
OXCT1
3-OXOACID COA TRANSFERASE 1




C) AnCg




Probe_ID (BPDv
Hs.14945_at
−4.158983584
201
R17684
Hs.14945
23305
ACSL6
ACYL-COA SYNTHETASE LONG-




C) AnCg







CHAIN FAMILY MEMBER 6




Probe_ID (BPDv
Hs.511880_at
−4.005278821
239
NM_000498
Hs.511880
1585
CYP11B2
CYTOCHROME P450, FAMILY 11,




C) AnCg







SUBFAMILY B, POLYPEPTIDE 2




Probe_ID (BPDv C) AnCg
Hs.149156_at
3.978596138
243
AI148899
Hs.149156
2731
GLDC
GLYCINE DEHYDROGENASE












(DECARBOXYLATING; GLYCINE












DECARBOXYLASE, GLYCINE












CLEAVAGE SYSTEM PROTEIN P)




Probe_ID (BPDv
Hs.433419_at
3.863300127
269
AA968887
Hs.433419
1327
COX4I1
CYTOCHROME C OXIDASE




C) AnCg







SUBUNIT IV ISOFORM 1




Probe_ID (BPDv
Hs.107476_at
3.852124446
271
AA961439
Hs.107476
10632
ATP5L
ATP SYNTHASE, H+




C) AnCg







TRANSPORTING,












MITOCHONDRIAL F0 COMPLEX,












SUBUNIT G




Probe_ID (BPDv
Hs.198269_at
3.834264322
274
AA970515
Hs.198269
4696
NDUFA3
NADH DEHYDROGENASE




C) AnCg







(UBIQUINONE) 1 ALPHA












SUBCOMPLEX, 3, 9 KDA




Probe_ID (BPDv
Hs.75859_at
3.825790421
277
AA975309
Hs.75859
740
MRPL49
MITOCHONDRIAL RIBOSOMAL




C) AnCg







PROTEIN L49




Probe_ID (BPDv
Hs.144130_at
−3.81230531
284
AA865892
Hs.144130
55186
FLJ10618
HYPOTHETICAL PROTEIN




C) AnCg







FLJ10618




Probe_ID (BPDv
Hs.3548_at
3.79318498
290
AA972489
Hs.3548
4515
MTCP1
MATURE T-CELL




C) AnCg







PROLIFERATION 1




Probe_ID (BPDv C) AnCg
Hs.7527_at
3.709781697
312
AA911794
Hs.7527
25996
DKFZP566E144
SMALL FRAGMENT NUCLEASE




Probe_ID (BPDv
Hs.154672_at
−3.613394429
338
AA962299
Hs.154672
10797
MTHFD2
METHYLENE




C) AnCg







TETRAHYDROFOLATE












DEHYDROGENASE (NAD+












DEPENDENT),












METHENYLTETRAHYDROFOLATE












CYCLOHYDROLASE




Probe_ID (BPDv
Hs.151573_at
3.581789697
347
AA905519
Hs.151573
1407
CRY1
CRYPTOCHROME 1




C) AnCg







(PHOTOLYASE-LIKE)




Probe_ID (BPDv
Hs.384944_at
−3.45700168
390
R15701
Hs.384944
6648
SOD2
SUPEROXIDE DISMUTASE 2,




C) AnCg







MITOCHONDRIAL




Probe_ID (BPDv
Hs.8364_at
3.403571903
412
AI051630
Hs.8364
5166
PDK4
PYRUVATE DEHYDROGENASE




C) AnCg







KINASE, ISOENZYME 4




Probe_ID (BPDv C) AnCg
Hs.139410_at
−3.380539628
424
AI004719
Hs.139410
1629
DBT
DIHYDROLIPOAMIDE BRANCHED












CHAIN TRANSACYLASE (E2












COMPONENT OF BRANCHED












CHAIN KETO ACID












DEHYDROGENASE COMPLEX;












MAPLE SYRUP URINE DISEASE)




Probe_ID (BPDv
Hs.409430_at
3.374251626
427
AA954940
Hs.409430
1716
DGUOK
DEOXYGUANOSINE KINASE




C) AnCg




Probe_ID (BPDv
Hs.300463_at
3.35163141
436

Hs.300463




C) AnCg




Probe_ID (BPDv
Hs.211914_at
3.322183756
446
R15290
Hs.211914
374291
NDUFS7
NADH DEHYDROGENASE




C) AnCg







(UBIQUINONE) FE-S PROTEIN 7,












20 KDA (NADH-COENZYME Q












REDUCTASE)




Probe_ID (MDD v
Hs.3260_at
−8.020507698
35
AA972181
Hs.3260
5663
PSEN1
PRESENILIN 1 (ALZHEIMER




C) AnCg







DISEASE 3)




Probe_ID (MDD v
Hs.109052_at
6.385329749
97
AA865834
Hs.109052
9556
C14orf2
CHROMOSOME 14 OPEN




C) AnCg







READING FRAME 2




Probe_ID (MDD v
Hs.75335_at
−5.88132842
127
AA888620
Hs.75335
145663
GATM
GLYCINE




C) AnCg







AMIDINOTRANSFERASE (L-












ARGININE:GLYCINE












AMIDINOTRANSFERASE)




Probe_ID (MDD v
Hs.247309_at
−5.878102056
128

Hs.247309




C) AnCg




Probe_ID (MDD v C) AnCg
Hs.433419_at
5.758749759
136
AA968887
Hs.433419
1327
COX4I1
CYTOCHROME C OXIDASE












SUBUNIT IV ISOFORM 1




Probe_ID (MDD v
Hs.1342_at
5.348334665
175
AA902930
Hs.1342
1329
COX5B
CYTOCHROME C OXIDASE




C) AnCg







SUBUNIT VB




Probe_ID (MDD v
Hs.49598_at
5.345843386
176
AA969459
Hs.49598
23600
AMACR
ALPHA-METHYLACYL-COA




C) AnCg







RACEMASE




Probe_ID (MDD v
Hs.353282_at
5.301845199
185

Hs.353282




C) AnCg




Probe_ID (MDD v
Hs.14945_at
−5.275227195
190
R17684
Hs.14945
23305
ACSL6
ACYL-COA SYNTHETASE LONG-




C) AnCg







CHAIN FAMILY MEMBER 6




Probe_ID (MDD v
Hs.436405_at
5.186131647
200
AA976445
Hs.436405
3420
IDH3B
ISOCITRATE DEHYDROGENASE




C) AnCg







3 (NAD+) BETA




Probe_ID (MDD v
Hs.20021_at
4.974117426
234
R17168
Hs.20021
6843
VAMP1
VESICLE-ASSOCIATED




C) AnCg







MEMBRANE PROTEIN 1












(SYNAPTOBREVIN 1)




Probe_ID (MDD v C) AnCg
Hs.182490_at
4.921605558
239
AA962471
Hs.182490
10128
LRPPRC
LEUCINE-RICH PPR-MOTIF












CONTAINING




Probe_ID (MDD v
Hs.153792_at
−4.902140124
243
AA905280
Hs.153792
4552
MTRR
5-METHYLTETRAHYDROFOLATE-




C) AnCg







HOMOCYSTEINE












METHYLTRANSFERASE












REDUCTASE




Probe_ID (MDD v
Hs.3100_at
4.804444009
257
AA954213
Hs.3100
3735
KARS
LYSYL-TRNA SYNTHETASE




C) AnCg




Probe_ID (MDD v
Hs.423404_at
4.802805566
259
AA975616
Hs.423404
9167
COX7A2L
CYTOCHROME C OXIDASE




C) AnCg







SUBUNIT VIIA POLYPEPTIDE 2












LIKE




Probe_ID (MDD v
Hs.279939_at
4.722208682
269
AA977776
Hs.279939
23787
MTCH1
MITOCHONDRIAL CARRIER




C) AnCg







HOMOLOG 1 (C. ELEGANS)




Probe_ID (MDD v C) AnCg
Hs.128410_at
4.581815327
287
AA886323
Hs.128410
2744
GLS
GLUTAMINASE




Probe_ID (MDD v
Hs.293970_at
−4.511696936
306
AA975064
Hs.293970
4329
ALDH6A1
ALDEHYDE DEHYDROGENASE 6




C) AnCg







FAMILY, MEMBER A1




Probe_ID (MDD v
Hs.528295_at
−4.499310703
310
AI022321
Hs.528295
10157
AASS
AMINOADIPATE-SEMIALDEHYDE




C) AnCg







SYNTHASE




Probe_ID (MDD v
Hs.75760_at
−4.363940471
336
R19294
Hs.75760
6342
SCP2
STEROL CARRIER PROTEIN 2




C) AnCg




Probe_ID (MDD v
Hs.9599_at
−4.285016072
355
AI032701
Hs.9599
10165
SLC25A13
SOLUTE CARRIER FAMILY 25,




C) AnCg







MEMBER 13 (CITRIN)




Probe_ID (MDD v
Hs.7744_at
4.058060577
409
AA954362
Hs.7744
4723
NDUFV1
NADH DEHYDROGENASE




C) AnCg







(UBIQUINONE) FLAVOPROTEIN 1,












51 KDA




Probe_ID (MDD v C) AnCg
Hs.350702_at
4.051168637
410
AA968881
Hs.350702
23479
ISCU
IRON-SULFUR CLUSTER












ASSEMBLY ENZYME




Probe_ID (MDD v
Hs.77690_at
−4.044194861
414
R14803
Hs.77690
5869
RAB5B
RAB5B, MEMBER RAS




C) AnCg







ONCOGENE FAMILY




Probe_ID (MDD v
Hs.287518_at
−3.967121349
437
AA970122
Hs.287518
5414
PNUTL2
PEANUT-LIKE 2 (DROSOPHILA)




C) AnCg




Probe_ID (MDD v
Hs.1342_at
8.751574623
7
AA902930
Hs.1342
1329
COX5B
CYTOCHROME C OXIDASE




C) DLPFC







SUBUNIT VB




Probe_ID (MDD v
Hs.182490_at
8.000187313
18
AA962471
Hs.182490
10128
LRPPRC
LEUCINE-RICH PPR-MOTIF




C) DLPFC







CONTAINING




Probe_ID (MDD v
Hs.128410_at
7.058759677
48
AA886323
Hs.128410
2744
GLS
GLUTAMINASE




C) DLPFC




Probe_ID (MDD v
Hs.353282_at
6.886768262
54

Hs.353282




C) DLPFC




Probe_ID (MDD v
Hs.173554_at
6.797907829
64
AA961851
Hs.173554
7385
UQCRC2
UBIQUINOL-CYTOCHROME C




C) DLPFC







REDUCTASE CORE PROTEIN II




Probe_ID (MDD v
Hs.405860_at
6.789034734
66
AA961135
Hs.405860
4191
MDH2
MALATE DEHYDROGENASE 2,




C) DLPFC







NAD (MITOCHONDRIAL)




Probe_ID (MDD v
Hs.3100_at
6.757315126
70
AA954213
Hs.3100
3735
KARS
LYSYL-TRNA SYNTHETASE




C) DLPFC




Probe_ID (MDD v C) DLPFC
Hs.505824_at
6.576861944
81
AI023273
Hs.505824
25813
CGI-51
CGI-51 PROTEIN




Probe_ID (MDD v
Hs.81886_at
6.233700436
98
AA905726
Hs.81886
549
AUH
AU RNA BINDING




C) DLPFC







PROTEIN/ENOYL-COENZYME A












HYDRATASE




Probe_ID (MDD v
Hs.279939_at
6.126403025
103
AA977776
Hs.279939
23787
MTCH1
MITOCHONDRIAL CARRIER




C) DLPFC







HOMOLOG 1 (C. ELEGANS)




Probe_ID (MDD v
Hs.290404_at
5.971266153
119
AA954185
Hs.290404
5250
SLC25A3
SOLUTE CARRIER FAMILY 25




C) DLPFC







(MITOCHONDRIAL CARRIER;












PHOSPHATE CARRIER), MEMBER 3




Probe_ID (MDD v
Hs.196270_at
5.880975299
124
AI126840
Hs.196270
81034
MFTC
MITOCHONDRIAL FOLATE




C) DLPFC







TRANSPORTER/CARRIER




Probe_ID (MDD v
Hs.350702_at
5.844647665
128
AA968881
Hs.350702
23479
ISCU
IRON-SULFUR CLUSTER




C) DLPFC







ASSEMBLY ENZYME




Probe_ID (MDD v C) DLPFC
Hs.62185_at
5.820175161
130
AA954490
Hs.62185
10479
SLC9A6
SOLUTE CARRIER FAMILY 9












(SODIUM/HYDROGEN












EXCHANGER), ISOFORM 6




Probe_ID (MDD v
Hs.194673_at
5.818156637
131
AA954646
Hs.194673
8682
PEA15
PHOSPHOPROTEIN ENRICHED IN




C) DLPFC







ASTROCYTES 15




Probe_ID (MDD v
Hs.439510_at
5.725481617
147
AA989107
Hs.439510
10730
YME1L1
YME1-LIKE 1 (S. CEREVISIAE)




C) DLPFC




Probe_ID (MDD v
Hs.109052_at
5.710330465
150
AA865834
Hs.109052
9556
C14orf2
CHROMOSOME 14 OPEN




C) DLPFC







READING FRAME 2




Probe_ID (MDD v
Hs.409140_at
5.639633997
156
AA864297
Hs.409140
539
ATP5O
ATP SYNTHASE, H+




C) DLPFC







TRANSPORTING,












MITOCHONDRIAL F1 COMPLEX,












O SUBUNIT (OLIGOMYCIN












SENSITIVITY CONFERRING












PROTEIN)




Probe_ID (MDD v
Hs.250616_at
5.60246391
159

Hs.250616




C) DLPFC




Probe_ID (MDD v
Hs.182217_at
5.57828738
164
AA984684
Hs.182217
8803
SUCLA2
SUCCINATE-COA LIGASE, ADP-




C) DLPFC







FORMING, BETA SUBUNIT




Probe_ID (MDD v
Hs.7744_at
5.54184827
169
AA954362
Hs.7744
4723
NDUFV1
NADH DEHYDROGENASE




C) DLPFC







(UBIQUINONE) FLAVOPROTEIN 1,












51 KDA




Probe_ID (MDD v
Hs.436405_at
5.39739172
196
AA976445
Hs.436405
3420
IDH3B
ISOCITRATE DEHYDROGENASE




C) DLPFC







3 (NAD+) BETA




Probe_ID (MDD v
Hs.126608_at
5.3276937
207
AA962235
Hs.126608
2908
NR3C1
NUCLEAR RECEPTOR




C) DLPFC







SUBFAMILY 3, GROUP C,












MEMBER 1 (GLUCOCORTICOID












RECEPTOR)




Probe_ID (MDD v
Hs.2043_at
5.280565636
214
AA977341
Hs.2043
291
SLC25A4
SOLUTE CARRIER FAMILY 25




C) DLPFC







(MITOCHONDRIAL CARRIER;












ADENINE NUCLEOTIDE












TRANSLOCATOR), MEMBER 4




Probe_ID (MDD v
Hs.144130_at
5.2188642
224
AA865892
Hs.144130
55186
FLJ10618
HYPOTHETICAL PROTEIN




C) DLPFC







FLJ10618




Probe_ID (MDD v
Hs.78614_at
5.189788976
230

Hs.78614




C) DLPFC




Probe_ID (MDD v
Hs.20716_at
5.188149858
231
AI021900
Hs.20716
10440
TIMM17A
TRANSLOCASE OF INNER




C) DLPFC







MITOCHONDRIAL MEMBRANE 17












HOMOLOG A (YEAST)




Probe_ID (MDD v
Hs.407860_at
5.099673885
245
AA973726
Hs.407860
4718
NDUFC2
NADH DEHYDROGENASE




C) DLPFC







(UBIQUINONE) 1, SUBCOMPLEX












UNKNOWN, 2, 14.5 KDA




Probe_ID (MDD v
Hs.20021_at
5.093990486
246
R17168
Hs.20021
6843
VAMP1
VESICLE-ASSOCIATED




C) DLPFC







MEMBRANE PROTEIN 1












(SYNAPTOBREVIN 1)




Probe_ID (MDD v
Hs.268016_at
5.077587148
250
AA987838
Hs.268016
64968
MRPS6
MITOCHONDRIAL RIBOSOMAL




C) DLPFC







PROTEIN S6




Probe_ID (MDD v
Hs.44298_at
5.017621789
266
AI141870
Hs.44298
51373
MRPS17
MITOCHONDRIAL RIBOSOMAL




C) DLPFC







PROTEIN S17




Probe_ID (MDD v
Hs.19236_at
4.976913834
281
AA888160
Hs.19236
4711
NDUFB5
NADH DEHYDROGENASE




C) DLPFC







(UBIQUINONE) 1 BETA












SUBCOMPLEX, 5, 16 KDA




Probe_ID (MDD v
Hs.429_at
4.87511346
311
BI761550
Hs.429
518
ATP5G3
ATP SYNTHASE, H+




C) DLPFC







TRANSPORTING,












MITOCHONDRIAL F0 COMPLEX,












SUBUNIT C (SUBUNIT 9)












ISOFORM 3




Probe_ID (MDD v
Hs.131273_at
4.855540013
318
AA978176
Hs.131273
4976
OPA1
OPTIC ATROPHY 1 (AUTOSOMAL




C) DLPFC







DOMINANT)




Probe_ID (MDD v
Hs.433419_at
4.837055096
325
AA968887
Hs.433419
1327
COX4I1
CYTOCHROME C OXIDASE




C) DLPFC







SUBUNIT IV ISOFORM 1




Probe_ID (MDD v
Hs.436988_at
4.817176809
332
AI025620
Hs.436988
1353
COX11
COX11 HOMOLOG,




C) DLPFC







CYTOCHROME C OXIDASE












ASSEMBLY PROTEIN (YEAST)




Probe_ID (MDD v
Hs.119251_at
4.800384591
337
AA954171
Hs.119251
7384
UQCRC1
UBIQUINOL-CYTOCHROME C




C) DLPFC







REDUCTASE CORE PROTEIN I




Probe_ID (MDD v
Hs.37_at
4.766384912
340
AI139512
Hs.37
38
ACAT1
ACETYL-COENZYME A




C) DLPFC







ACETYLTRANSFERASE 1












(ACETOACETYL COENZYME A












THIOLASE)




Probe_ID (MDD v
Hs.11866_at
4.718266874
353
AA974086
Hs.11866
10431
TIMM23
TRANSLOCASE OF INNER




C) DLPFC







MITOCHONDRIAL MEMBRANE 23












HOMOLOG (YEAST)




Probe_ID (MDD v
Hs.444757_at
4.716048741
354
AA968467
Hs.444757
23095
KIF1B
KINESIN FAMILY MEMBER 1B




C) DLPFC




Probe_ID (MDD v
Hs.5556_at
4.68842541
358
AA961682
Hs.5556
4706
NDUFAB1
NADH DEHYDROGENASE




C) DLPFC







(UBIQUINONE) 1, ALPHA/BETA












SUBCOMPLEX, 1, 8 KDA




Probe_ID (MDD v
Hs.211571_at
4.682127722
362
AA923565
Hs.211571
3052
HCCS
HOLOCYTOCHROME C




C) DLPFC







SYNTHASE (CYTOCHROME C












HEME-LYASE)




Probe_ID (MDD v
Hs.289271_at
4.679567034
365
AA863409
Hs.289271
1537
CYC1
CYTOCHROME C-1




C) DLPFC




Probe_ID (MDD v
Hs.184860_at
4.647024454
373
AA973406
Hs.184860
57128
C6orf149
CHROMOSOME 6 OPEN




C) DLPFC







READING FRAME 149




Probe_ID (MDD v
Hs.247309_at
−4.631146258
374

Hs.247309




C) DLPFC




Probe_ID (MDD v
Hs.76366_at
4.541868883
388
AA999694
Hs.76366
572
BAD
BCL2-ANTAGONIST OF CELL




C) DLPFC







DEATH




Probe_ID (MDD v
Hs.184233_at
4.524597014
395
AA961767
Hs.184233
3313
HSPA9B
HEAT SHOCK 70 KDA PROTEIN 9B




C) DLPFC







(MORTALIN-2)




Probe_ID (MDD v
Hs.300463_at
4.491205579
403

Hs.300463




C) DLPFC




Probe_ID (MDD v
Hs.107476_at
4.487968916
405
AA961439
Hs.107476
10632
ATP5L
ATP SYNTHASE, H+




C) DLPFC







TRANSPORTING,












MITOCHONDRIAL F0 COMPLEX,












SUBUNIT G




Probe_ID (MDD v
Hs.43549_at
4.482655426
407
AA975374
Hs.43549
55847
C10orf70
CHROMOSOME 10 OPEN




C) DLPFC







READING FRAME 70




Probe_ID (MDD v
Hs.161357_at
4.415481565
415
R14727
Hs.161357
5162
PDHB
PYRUVATE DEHYDROGENASE




C) DLPFC







(LIPOAMIDE) BETA




Probe_ID (MDD v
Hs.198269_at
4.313217236
434
AA970515
Hs.198269
4696
NDUFA3
NADH DEHYDROGENASE




C) DLPFC







(UBIQUINONE) 1 ALPHA












SUBCOMPLEX, 3, 9 KDA







Summary MDD with Drecti Change

















0
2
Probe_ID
Hs.107476_at
3.852124446
271
AA961439
Hs.107476
10632
ATP5L
ATP SYNTHASE, H+




(BPDv C)







TRANSPORTING,




AnCg







MITOCHONDRIAL F0 COMPLEX,












SUBUNIT G


1
3
Probe_ID
Hs.107476_at
4.487968916
405
AA961439
Hs.107476
10632
ATP5L
ATP SYNTHASE, H+




(MDD v







TRANSPORTING,




C)







MITOCHONDRIAL F0 COMPLEX,




DLPFC







SUBUNIT G


0
4
Probe_ID
Hs.109052_at
5.019252834
103
AA865834
Hs.109052
9556
C14orf2
CHROMOSOME 14 OPEN




(BPDv C)







READING FRAME 2




AnCg


0
5
Probe_ID
Hs.109052_at
6.385329749
97
AA865834
Hs.109052
9556
C14orf2
CHROMOSOME 14 OPEN




(MDD v







READING FRAME 2




C) AnCg


1
6
Probe_ID
Hs.109052_at
5.710330465
150
AA865834
Hs.109052
9556
C14orf2
CHROMOSOME 14 OPEN




(MDD v







READING FRAME 2




C)




DLPFC


1
7
Probe_ID
Hs.11866_at
4.718266874
353
AA974086
Hs.11866
10431
TIMM23
TRANSLOCASE OF INNER




(MDD v







MITOCHONDRIAL MEMBRANE 23




C)







HOMOLOG (YEAST)




DLPFC


1
8
Probe_ID
Hs.119251_at
4.800384591
337
AA954171
Hs.119251
7384
UQCRC1
UBIQUINOL-CYTOCHROME C




(MDD v







REDUCTASE CORE PROTEIN I




C)




DLPFC


1
9
Probe_ID
Hs.119316_at
7.019916677
17
AI075114
Hs.119316
5188
PET112L
PET112-LIKE (YEAST)




(BPDv C)




AnCg


1
10
Probe_ID
Hs.126608_at
5.3276937
207
AA962235
Hs.126608
2908
NR3C1
NUCLEAR RECEPTOR




(MDD v







SUBFAMILY 3, GROUP C,




C)







MEMBER 1 (GLUCOCORTICOID




DLPFC







RECEPTOR)


0
11
Probe_ID
Hs.128410_at
4.581815327
287
AA886323
Hs.128410
2744
GLS
GLUTAMINASE




(MDD v




C) AnCg


1
12
Probe_ID
Hs.128410_at
7.058759677
48
AA886323
Hs.128410
2744
GLS
GLUTAMINASE




(MDD v




C)




DLPFC


1
13
Probe_ID
Hs.131273_at
4.855540013
318
AA978176
Hs.131273
4976
OPA1
OPTIC ATROPHY 1 (AUTOSOMAL




(MDD v







DOMINANT)




C)




DLPFC


0
14
Probe_ID
Hs.1342_at
5.348334665
175
AA902930
Hs.1342
1329
COX5B
CYTOCHROME C OXIDASE




(MDD v







SUBUNIT VB




C) AnCg


1
15
Probe_ID
Hs.1342_at
8.751574623
7
AA902930
Hs.1342
1329
COX5B
CYTOCHROME C OXIDASE




(MDD v







SUBUNIT VB




C)




DLPFC


1
16
Probe_ID
Hs.139410_at
−3.380539628
424
AI004719
Hs.139410
1629
DBT
DIHYDROLIPOAMIDE BRANCHED




(BPDv C)







CHAIN TRANSACYLASE (E2




AnCg







COMPONENT OF BRANCHED












CHAIN KETO ACID












DEHYDROGENASE COMPLEX;












MAPLE SYRUP URINE DISEASE)


0
17
Probe_ID
Hs.144130_at
−3.81230531
284
AA865892
Hs.144130
55186
FLJ10618
HYPOTHETICAL PROTEIN




(BPDv C)







FLJ10618




AnCg


1
18
Probe_ID
Hs.144130_at
5.2188642
224
AA865892
Hs.144130
55186
FLJ10618
HYPOTHETICAL PROTEIN




(MDD v







FLJ10618




C)




DLPFC


1
19
Probe_ID
Hs.149156_at
3.978596138
243
AI148899
Hs.149156
2731
GLDC
GLYCINE DEHYDROGENASE




(BPDv C)







(DECARBOXYLATING; GLYCINE




AnCg







DECARBOXYLASE, GLYCINE












CLEAVAGE SYSTEM PROTEIN P)


0
20
Probe_ID
Hs.14945_at
−4.158983584
201
R17684
Hs.14945
23305
ACSL6
ACYL-COA SYNTHETASE LONG-




(BPDv C)







CHAIN FAMILY MEMBER 6




AnCg


1
21
Probe_ID
Hs.14945_at
−5.275227195
190
R17684
Hs.14945
23305
ACSL6
ACYL-COA SYNTHETASE LONG-




(MDD v







CHAIN FAMILY MEMBER 6




C) AnCg


1
22
Probe_ID
Hs.151573_at
3.581789697
347
AA905519
Hs.151573
1407
CRY1
CRYPTOCHROME 1




(BPDv C)







(PHOTOLYASE-LIKE)




AnCg


1
23
Probe_ID
Hs.153792_at
−4.902140124
243
AA905280
Hs.153792
4552
MTRR
5-METHYLTETRAHYDROFOLATE-




(MDD v







HOMOCYSTEINE




C) AnCg







METHYLTRANSFERASE












REDUCTASE


1
24
Probe_ID
Hs.154672_at
−3.613394429
338
AA962299
Hs.154672
10797
MTHFD2
METHYLENE




(BPDv C)







TETRAHYDROFOLATE




AnCg







DEHYDROGENASE (NAD+












DEPENDENT),












METHENYLTETRAHYDROFOLATE












CYCLOHYDROLASE


1
25
Probe_ID
Hs.161357_at
4.415481565
415
R14727
Hs.161357
5162
PDHB
PYRUVATE DEHYDROGENASE




(MDD v







(LIPOAMIDE) BETA




C)




DLPFC


1
26
Probe_ID
Hs.173554_at
6.797907829
64
AA961851
Hs.173554
7385
UQCRC2
UBIQUINOL-CYTOCHROME C




(MDD v







REDUCTASE CORE PROTEIN II




C)




DLPFC


1
27
Probe_ID
Hs.177584_at
4.165654802
200
AA961910
Hs.177584
5019
OXCT1
3-OXOACID COA TRANSFERASE 1




(BPDv C)




AnCg


1
28
Probe_ID
Hs.182217_at
5.57828738
164
AA984684
Hs.182217
8803
SUCLA2
SUCCINATE-COA LIGASE, ADP-




(MDD v







FORMING, BETA SUBUNIT




C)




DLPFC


0
29
Probe_ID
Hs.182490_at
4.921605558
239
AA962471
Hs.182490
10128
LRPPRC
LEUCINE-RICH PPR-MOTIF




(MDD v







CONTAINING




C) AnCg


1
30
Probe_ID
Hs.182490_at
8.000187313
18
AA962471
Hs.182490
10128
LRPPRC
LEUCINE-RICH PPR-MOTIF




(MDD v







CONTAINING




C)




DLPFC


1
31
Probe_ID
Hs.184233_at
4.524597014
395
AA961767
Hs.184233
3313
HSPA9B
HEAT SHOCK 70 KDA PROTEIN 9B




(MDD v







(MORTALIN-2)




C)




DLPFC


1
32
Probe_ID
Hs.184860_at
4.647024454
373
AA973406
Hs.184860
57128
C6orf149
CHROMOSOME 6 OPEN




(MDD v







READING FRAME 149




C)




DLPFC


1
33
Probe_ID
Hs.19236_at
4.976913834
281
AA888160
Hs.19236
4711
NDUFB5
NADH DEHYDROGENASE




(MDD v







(UBIQUINONE) 1 BETA




C)







SUBCOMPLEX, 5, 16 KDA




DLPFC


0
34
Probe_ID
Hs.194673_at
6.739939746
24
AA954646
Hs.194673
8682
PEA15
PHOSPHOPROTEIN ENRICHED IN




(BPDv C)







ASTROCYTES 15




AnCg


1
35
Probe_ID
Hs.194673_at
5.818156637
131
AA954646
Hs.194673
8682
PEA15
PHOSPHOPROTEIN ENRICHED IN




(MDD v







ASTROCYTES 15




C)




DLPFC


1
36
Probe_ID
Hs.196270_at
5.880975299
124
AI126840
Hs.196270
81034
MFTC
MITOCHONDRIAL FOLATE




(MDD v







TRANSPORTER/CARRIER




C)




DLPFC


0
37
Probe_ID
Hs.198269_at
3.834264322
274
AA970515
Hs.198269
4696
NDUFA3
NADH DEHYDROGENASE




(BPDv C)







(UBIQUINONE) 1 ALPHA




AnCg







SUBCOMPLEX, 3, 9 KDA


1
38
Probe_ID
Hs.198269_at
4.313217236
434
AA970515
Hs.198269
4696
NDUFA3
NADH DEHYDROGENASE




(MDD v







(UBIQUINONE) 1 ALPHA




C)







SUBCOMPLEX, 3, 9 KDA




DLPFC


0
39
Probe_ID
Hs.20021_at
−7.727057737
9
R17168
Hs.20021
6843
VAMP1
VESICLE-ASSOCIATED




(BPDv C)







MEMBRANE PROTEIN 1




AnCg







(SYNAPTOBREVIN 1)


0
40
Probe_ID
Hs.20021_at
4.974117426
234
R17168
Hs.20021
6843
VAMP1
VESICLE-ASSOCIATED




(MDD v







MEMBRANE PROTEIN 1




C) AnCg







(SYNAPTOBREVIN 1)


1
41
Probe_ID
Hs.20021_at
5.093990486
246
R17168
Hs.20021
6843
VAMP1
VESICLE-ASSOCIATED




(MDD v







MEMBRANE PROTEIN 1




C)







(SYNAPTOBREVIN 1)




DLPFC


1
42
Probe_ID
Hs.2043_at
5.280565636
214
AA977341
Hs.2043
291
SLC25A4
SOLUTE CARRIER FAMILY 25




(MDD v







(MITOCHONDRIAL CARRIER;




C)







ADENINE NUCLEOTIDE




DLPFC







TRANSLOCATOR), MEMBER 4


1
43
Probe_ID
Hs.20716_at
5.188149858
231
AI021900
Hs.20716
10440
TIMM17A
TRANSLOCASE OF INNER




(MDD v







MITOCHONDRIAL MEMBRANE 17




C)







HOMOLOG A (YEAST)




DLPFC


1
44
Probe_ID
Hs.211571_at
4.682127722
362
AA923565
Hs.211571
3052
HCCS
HOLOCYTOCHROME C




(MDD v







SYNTHASE (CYTOCHROME C




C)







HEME-LYASE)




DLPFC


1
45
Probe_ID
Hs.211914_at
3.322183756
446
R15290
Hs.211914
374291
NDUFS7
NADH DEHYDROGENASE




(BPDv C)







(UBIQUINONE) FE-S PROTEIN 7,




AnCg







20 KDA (NADH-COENZYME Q












REDUCTASE)


1
46
Probe_ID
Hs.243491_at
−4.224934266
183

Hs.243491




(BPDv C)




AnCg


0
47
Probe_ID
Hs.247309_at
−5.878102056
128

Hs.247309




(MDD v




C) AnCg


1
48
Probe_ID
Hs.247309_at
−4.631146258
374

Hs.247309




(MDD v




C)




DLPFC


1
49
Probe_ID
Hs.250616_at
5.60246391
159

Hs.250616




(MDD v




C)




DLPFC


1
50
Probe_ID
Hs.268016_at
5.077587148
250
AA987838
Hs.268016
64968
MRPS6
MITOCHONDRIAL RIBOSOMAL




(MDD v







PROTEIN S6




C)




DLPFC


0
51
Probe_ID
Hs.279939_at
4.582420323
138
AA977776
Hs.279939
23787
MTCH1
MITOCHONDRIAL CARRIER




(BPDv C)







HOMOLOG 1 (C. ELEGANS)




AnCg


0
52
Probe_ID
Hs.279939_at
4.722208682
269
AA977776
Hs.279939
23787
MTCH1
MITOCHONDRIAL CARRIER




(MDD v







HOMOLOG 1 (C. ELEGANS)




C) AnCg


1
53
Probe_ID
Hs.279939_at
6.126403025
103
AA977776
Hs.279939
23787
MTCH1
MITOCHONDRIAL CARRIER




(MDD v







HOMOLOG 1 (C. ELEGANS)




C)




DLPFC


1
54
Probe_ID
Hs.287518_at
−3.967121349
437
AA970122
Hs.287518
5414
PNUTL2
PEANUT-LIKE 2 (DROSOPHILA)




(MDD v




C) AnCg


1
55
Probe_ID
Hs.289271_at
4.679567034
365
AA863409
Hs.289271
1537
CYC1
CYTOCHROME C-1




(MDD v




C)




DLPFC


1
56
Probe_ID
Hs.290404_at
5.971266153
119
AA954185
Hs.290404
5250
SLC25A3
SOLUTE CARRIER FAMILY 25




(MDD v







(MITOCHONDRIAL CARRIER;




C)







PHOSPHATE CARRIER), MEMBER 3




DLPFC


1
57
Probe_ID
Hs.293970_at
−4.511696936
306
AA975064
Hs.293970
4329
ALDH6A1
ALDEHYDE DEHYDROGENASE 6




(MDD v







FAMILY, MEMBER A1




C) AnCg


0
58
Probe_ID
Hs.300463_at
3.35163141
436

Hs.300463




(BPDv C)




AnCg


1
59
Probe_ID
Hs.300463_at
4.491205579
403

Hs.300463




(MDD v




C)




DLPFC


0
60
Probe_ID
Hs.3100_at
4.309487236
169
AA954213
Hs.3100
3735
KARS
LYSYL-TRNA SYNTHETASE




(BPDv C)




AnCg


0
61
Probe_ID
Hs.3100_at
4.804444009
257
AA954213
Hs.3100
3735
KARS
LYSYL-TRNA SYNTHETASE




(MDD v




C) AnCg


1
62
Probe_ID
Hs.3100_at
6.757315126
70
AA954213
Hs.3100
3735
KARS
LYSYL-TRNA SYNTHETASE




(MDD v




C)




DLPFC


1
63
Probe_ID
Hs.3254_at
4.193592918
195
AA971301
Hs.3254
6150
MRPL23
MITOCHONDRIAL RIBOSOMAL




(BPDv C)







PROTEIN L23




AnCg


1
64
Probe_ID
Hs.3260_at
−8.020507698
35
AA972181
Hs.3260
5663
PSEN1
PRESENILIN 1 (ALZHEIMER




(MDD v







DISEASE 3)




C) AnCg


0
65
Probe_ID
Hs.350702_at
4.051168637
410
AA968881
Hs.350702
23479
ISCU
IRON-SULFUR CLUSTER




(MDD v







ASSEMBLY ENZYME




C) AnCg


1
66
Probe_ID
Hs.350702_at
5.844647665
128
AA968881
Hs.350702
23479
ISCU
IRON-SULFUR CLUSTER




(MDD v







ASSEMBLY ENZYME




C)




DLPFC


0
67
Probe_ID
Hs.353282_at
5.301845199
185

Hs.353282




(MDD v




C) AnCg


1
68
Probe_ID
Hs.353282_at
6.886768262
54

Hs.353282




(MDD v




C)




DLPFC


1
69
Probe_ID
Hs.3548_at
3.79318498
290
AA972489
Hs.3548
4515
MTCP1
MATURE T-CELL




(BPDv C)







PROLIFERATION 1




AnCg


1
70
Probe_ID
Hs.356231_at
−6.670345597
28
AI038963
Hs.356231
79085
SLC25A23
SOLUTE CARRIER FAMILY 25




(BPDv C)







(MITOCHONDRIAL CARRIER;




AnCg







PHOSPHATE CARRIER), MEMBER












23


1
71
Probe_ID
Hs.37_at
4.766384912
340
AI139512
Hs.37
38
ACAT1
ACETYL-COENZYME A




(MDD v







ACETYLTRANSFERASE 1




C)







(ACETOACETYL COENZYME A




DLPFC







THIOLASE)


1
72
Probe_ID
Hs.384944_at
−3.45700168
390
R15701
Hs.384944
6648
SOD2
SUPEROXIDE DISMUTASE 2,




(BPDv C)







MITOCHONDRIAL




AnCg


1
73
Probe_ID
Hs.405860_at
6.789034734
66
AA961135
Hs.405860
4191
MDH2
MALATE DEHYDROGENASE 2,




(MDD v







NAD (MITOCHONDRIAL)




C)




DLPFC


1
74
Probe_ID
Hs.407860_at
5.099673885
245
AA973726
Hs.407860
4718
NDUFC2
NADH DEHYDROGENASE




(MDD v







(UBIQUINONE) 1, SUBCOMPLEX




C)







UNKNOWN, 2, 14.5 KDA




DLPFC


1
75
Probe_ID
Hs.409140_at
5.639633997
156
AA864297
Hs.409140
539
ATP5O
ATP SYNTHASE, H+




(MDD v







TRANSPORTING,




C)







MITOCHONDRIAL F1 COMPLEX,




DLPFC







O SUBUNIT (OLIGOMYCIN












SENSITIVITY CONFERRING












PROTEIN)


1
76
Probe_ID
Hs.409430_at
3.374251626
427
AA954940
Hs.409430
1716
DGUOK
DEOXYGUANOSINE KINASE




(BPDv C)




AnCg


1
77
Probe_ID
Hs.423404_at
4.802805566
259
AA975616
Hs.423404
9167
COX7A2L
CYTOCHROME C OXIDASE




(MDD v







SUBUNIT VIIA POLYPEPTIDE 2




C) AnCg







LIKE


1
78
Probe_ID
Hs.429_at
4.87511346
311
BI761550
Hs.429
518
ATP5G3
ATP SYNTHASE, H+




(MDD v







TRANSPORTING,




C)







MITOCHONDRIAL F0 COMPLEX,




DLPFC







SUBUNIT C (SUBUNIT 9)












ISOFORM 3


0
79
Probe_ID
Hs.433419_at
3.863300127
269
AA968887
Hs.433419
1327
COX4I1
CYTOCHROME C OXIDASE




(BPDv C)







SUBUNIT IV ISOFORM 1




AnCg


0
80
Probe_ID
Hs.433419_at
5.758749759
136
AA968887
Hs.433419
1327
COX4I1
CYTOCHROME C OXIDASE




(MDD v







SUBUNIT IV ISOFORM 1




C) AnCg


1
81
Probe_ID
Hs.433419_at
4.837055096
325
AA968887
Hs.433419
1327
COX4I1
CYTOCHROME C OXIDASE




(MDD v







SUBUNIT IV ISOFORM 1




C)




DLPFC


1
82
Probe_ID
Hs.43549_at
4.482655426
407
AA975374
Hs.43549
55847
C10orf70
CHROMOSOME 10 OPEN




(MDD v







READING FRAME 70




C)




DLPFC


0
83
Probe_ID
Hs.436405_at
5.186131647
200
AA976445
Hs.436405
3420
IDH3B
ISOCITRATE DEHYDROGENASE




(MDD v







3 (NAD+) BETA




C) AnCg


1
84
Probe_ID
Hs.436405_at
5.39739172
196
AA976445
Hs.436405
3420
IDH3B
ISOCITRATE DEHYDROGENASE




(MDD v







3 (NAD+) BETA




C)




DLPFC


1
85
Probe_ID
Hs.436988_at
4.817176809
332
AI025620
Hs.436988
1353
COX11
COX11 HOMOLOG,




(MDD v







CYTOCHROME C OXIDASE




C)







ASSEMBLY PROTEIN (YEAST)




DLPFC


1
86
Probe_ID
Hs.438303_at
5.313202083
72
AA969386
Hs.438303
23395
LARS2
LEUCYL-TRNA SYNTHETASE 2,




(BPDv C)







MITOCHONDRIAL




AnCg


1
87
Probe_ID
Hs.439510_at
5.725481617
147
AA989107
Hs.439510
10730
YME1L1
YME1-LIKE 1 (S. CEREVISIAE)




(MDD v




C)




DLPFC


1
88
Probe_ID
Hs.44298_at
5.017621789
266
AI141870
Hs.44298
51373
MRPS17
MITOCHONDRIAL RIBOSOMAL




(MDD v







PROTEIN S17




C)




DLPFC


1
89
Probe_ID
Hs.444757_at
4.716048741
354
AA968467
Hs.444757
23095
KIF1B
KINESIN FAMILY MEMBER 1B




(MDD v




C)




DLPFC


1
90
Probe_ID
Hs.49598_at
5.345843386
176
AA969459
Hs.49598
23600
AMACR
ALPHA-METHYLACYL-COA




(MDD v







RACEMASE




C) AnCg


1
91
Probe_ID
Hs.505824_at
6.576861944
81
AI023273
Hs.505824
25813
CGI-51
CGI-51 PROTEIN




(MDD v




C)




DLPFC


1
92
Probe_ID
Hs.511880_at
−4.005278821
239
NM_000498
Hs.511880
1585
CYP11B2
CYTOCHROME P450, FAMILY 11,




(BPDv C)







SUBFAMILY B, POLYPEPTIDE 2




AnCg


1
93
Probe_ID
Hs.528295_at
−4.499310703
310
AI022321
Hs.528295
10157
AASS
AMINOADIPATE-SEMIALDEHYDE




(MDD v







SYNTHASE




C) AnCg


1
94
Probe_ID
Hs.5556_at
4.68842541
358
AA961682
Hs.5556
4706
NDUFAB1
NADH DEHYDROGENASE




(MDD v







(UBIQUINONE) 1, ALPHA/BETA




C)







SUBCOMPLEX, 1, 8 KDA




DLPFC


1
95
Probe_ID
Hs.62185_at
5.820175161
130
AA954490
Hs.62185
10479
SLC9A6
SOLUTE CARRIER FAMILY 9




(MDD v







(SODIUM/HYDROGEN




C)







EXCHANGER), ISOFORM 6




DLPFC


1
96
Probe_ID
Hs.74047_at
4.496465885
150

Hs.74047




(BPDv C)




AnCg


1
97
Probe_ID
Hs.7527_at
3.709781697
312
AA911794
Hs.7527
25996
DKFZP566E144
SMALL FRAGMENT NUCLEASE




(BPDv C)




AnCg


1
98
Probe_ID
Hs.75335_at
−5.88132842
127
AA888620
Hs.75335
145663
GATM
GLYCINE




(MDD v







AMIDINOTRANSFERASE (L-




C) AnCg







ARGININE: GLYCINE












AMIDINOTRANSFERASE)


1
99
Probe_ID
Hs.75760_at
−4.363940471
336
R19294
Hs.75760
6342
SCP2
STEROL CARRIER PROTEIN 2




(MDD v




C) AnCg


1
100
Probe_ID
Hs.75859_at
3.825790421
277
AA975309
Hs.75859
740
MRPL49
MITOCHONDRIAL RIBOSOMAL




(BPDv C)







PROTEIN L49




AnCg


0
101
Probe_ID
Hs.76366_at
4.693419228
127
AA999694
Hs.76366
572
BAD
BCL2-ANTAGONIST OF CELL




(BPDv C)







DEATH




AnCg


1
102
Probe_ID
Hs.76366_at
4.541868883
388
AA999694
Hs.76366
572
BAD
BCL2-ANTAGONIST OF CELL




(MDD v







DEATH




C)




DLPFC


0
103
Probe_ID
Hs.7744_at
4.058060577
409
AA954362
Hs.7744
4723
NDUFV1
NADH DEHYDROGENASE




(MDD v







(UBIQUINONE) FLAVOPROTEIN 1,




C) AnCg







51 KDA


1
104
Probe_ID
Hs.7744_at
5.54184827
169
AA954362
Hs.7744
4723
NDUFV1
NADH DEHYDROGENASE




(MDD v







(UBIQUINONE) FLAVOPROTEIN 1,




C)







51 KDA




DLPFC


1
105
Probe_ID
Hs.77690_at
−4.044194861
414
R14803
Hs.77690
5869
RAB5B
RAB5B, MEMBER RAS




(MDD v







ONCOGENE FAMILY




C) AnCg


1
106
Probe_ID
Hs.78614_at
5.189788976
230

Hs.78614




(MDD v




C)




DLPFC


1
107
Probe_ID
Hs.81886_at
6.233700436
98
AA905726
Hs.81886
549
AUH
AU RNA BINDING




(MDD v







PROTEIN/ENOYL-COENZYME A




C)







HYDRATASE




DLPFC


1
108
Probe_ID
Hs.8364_at
3.403571903
412
AI051630
Hs.8364
5166
PDK4
PYRUVATE DEHYDROGENASE




(BPDv C)







KINASE, ISOENZYME 4




AnCg


1
109
Probe_ID
Hs.9599_at
−4.285016072
355
AI032701
Hs.9599
10165
SLC25A13
SOLUTE CARRIER FAMILY 25,




(MDD v







MEMBER 13 (CITRIN)




C) AnCg
















TABLE 11







Genes dysregulated in MD, BP, and schizophrenia














UniGene







1
ID
probe set
Acc
Name
Symbol
Direction of Change
















2
Hs.282878
Hs.282878_at
NM_021633
Kelch-like 12
KLHL12
DLPFC-BPD&MDD&SCZ-U






(Drosophila)


3
Hs.435039
Hs.435039_at
NM_014914
Trinucleotide repeat
CENTG2
DLPFC-BPD&MDD&SCZ-U






containing 17


4
Hs.530712
Hs.530712_at
NM_017917
Chromosome 14 open
C14orf10
DLPFC-BPD&MDD&SCZ-U






reading frame 10


5
Hs.56294
Hs.56294_at
NM_004794
RAB33A, member
RAB33A
DLPFC-BPD&MDD&SCZ-U






RAS oncogene family


6
Hs.21577
Hs.21577_at
NM_005701
RNA, U transporter 1
RNUT1
DLPFC-BPD&SCZ-U


7
Hs.274479
Hs.274479−_at
NM_197972
Non-metastatic cells 7,
NME7
DLPFC-BPD&SCZ-U






protein expressed in






(nucleoside-






diphosphate kinase)


8
Hs.368486
Hs.368486_at
NM_001649
Apical protein-like
APXL
DLPFC-BPD&SCZ-U






(Xenopus laevis)


9
Hs.468415
Hs.468415_at
NM_002643
Phosphatidylinositol
PIGF
DLPFC-BPD&SCZ-U






glycan, class F


10
Hs.471401
Hs.471401_at
NM_004328
BCS1-like (yeast)
BCS1L
DLPFC-BPD&SCZ-U


11
Hs.502145
Hs.502145_at
NM_006157
NEL-like 1 (chicken)
NELL1
DLPFC-BPD&SCZ-U


12
Hs.514036
Hs.514036_at
NM_006923
Stromal cell-derived
SDF2
DLPFC-BPD&SCZ-U






factor 2


13
Hs.532853
Hs.532853+_at
NM_004146
NADH dehydrogenase
NDUFB7
DLPFC-BPD&SCZ-U






(ubiquinone) 1 beta






subcomplex, 7, 18 kDa


14
Hs.111779
Hs.111779_at
NM_003118
Secreted protein,
SPARC
DLPFC-BPD&MDD&SCZ-D






acidic, cysteine-rich






(osteonectin)


15
Hs.171695
Hs.171695_at
NM_004417
Dual specificity
DUSP1
DLPFC-BPD&MDD&SCZ-D






phosphatase 1


16
Hs.212838
Hs.212838−_at
NM_000014
Alpha-2-macroglobulin
A2M
DLPFC-BPD&MDD&SCZ-D


17
Hs.34560
Hs.34560−_at
NM_005574
LIM domain only 2
LMO2
DLPFC-BPD&MDD&SCZ-D






(rhombotin-like 1)


18
Hs.347270
Hs.347270−_at
NM_033554
Major
HLA-
DLPFC-BPD&MDD&SCZ-D






histocompatibility
DPA1






complex, class II, DP






alpha 1


19
Hs.436568
Hs.436568_at
BC024272
CD74 antigen
CD74
DLPFC-BPD&MDD&SCZ-D






(invariant polypeptide






of major






histocompatibility






complex, class II






antigen-associated)


20
Hs.491582
Hs.491582_at
NM_000931
Plasminogen activator,
PLAT
DLPFC-BPD&MDD&SCZ-D






tissue


21
Hs.534115
Hs.534115_at
NM_006988
A disintegrin-like and
ADAMTS1
DLPFC-BPD&MDD&SCZ-D






metalloprotease






(reprolysin type) with






thrombospondin type 1






motif, 1


22
Hs.17109
Hs.17109_at
NM_004867
Integral membrane
ITM2A
DLPFC-BPD&SCZ-D






protein 2A


23
Hs.485130
Hs.485130_at
K01615
Major
HLA-
DLPFC-BPD&SCZ-D






histocompatibility
DPB1






complex, class II, DP






beta 1


24
Hs.504877
Hs.504877_at
X69549
Rho GDP dissociation
ARHGDIB
DLPFC-BPD&SCZ-D






inhibitor (GDI) beta


25
Hs.520048
Hs.520048_at
NM_019111
Major
HLA-DRA
DLPFC-BPD&SCZ-D






histocompatibility






complex, class II, DR






alpha
















TABLE 12







Genes dysregulated in MD, BP, and schizophrenia













Symbol
Name
UniGene ID
AnCg
DLPFC
CB
nAcc





PTGDS
Prostaglandin
Hs.446429


CB-D
nAcc-D


PLAT
Plasminogen activator, tissue
Hs.491582
AnCg-D
DLPFC-D


ADAMTS1
Disintegrin-like and metalloprotease
Hs.534115

DLPFC-D

nAcc-D


















TABLE 13









Bipolar



Disorder



U95Av2



Cohort A













UniGene
GDB



P
% Fold


ID
Accession #
Name
Symbol
Cytoband
Value
Change










Monoamine Metabolism













Hs.370408
AL390148
Catechol-O-
COMT
22q11.21–q11.23
<0.01
15.8




methyltransfera


Hs.46732
NM_000898
Monoamine
MAOB
Xp11.23
<0.01
15.9




oxidase B







Neuropeptide Ligand













Hs.1832
BF680552
Neuropeptide Y
NPY
7p15.1
<0.01*
22.1


Hs.12409
BI918626
Somatostatin
SST
3q28
<0.01*
29.0


Hs.1408
BC053866
Endothelin 3
EDN3
20q13.2–q13.3
<0.01
−12.3







GPCRs













Hs.519057
L07615
Neuropeptide Y
NPY1R
4q31.3–q32






receptor Y1


Hs.131138
NM_012344
Neurotensin
NTSR2
2p25.1






receptor 2


Hs.112621
BC041407
Metabotropic
GRM3
7q21.1–q21.2
<0.01*
33.4




Glutamate




receptor 3


Hs.88372
BC033742
Tachykinin
TACR2
10q11–q21






receptor 2


Hs.154210
BC018650
Endothelial
EDG1
1p21






differentiation G-




protein-coupled




receptor 1


Hs.126667
BC036034
Endothelial
EDG2
9q31.3
<0.05
24.1




differentiation G-




protein-coupled




receptor 2


Hs.82002
NM_000115
Endothelin
EDNRB
13q22






receptor type B


Hs.406094
BX649006
G protein-coupled
GPR37
7q31
<0.01*
53.1




receptor 37


Hs.513633
NM_201524
G protein-coupled
GPR56
16q13






receptor 56


Hs.148685
NM_016235
G protein-coupled
GPRC5B
16p12
<0.01*
38.4




receptor C-5-B


Hs.99195
XM_291111
G protein-coupled
GPR125
4p15.31






receptor 125







G protein and Regulators













Hs.134587
BC026326
G protein alpha
GNAI1
7q21






inhibiting




activity




polypeptide 1


Hs.24950
NM_003617
Regulator of G-
RGS5
1q23.1
<0.01
−19.0




protein




signalling 5


Hs.368733
AK094559
Regulator of G-
RGS20
8q12.1






protein




signalling 20







Cyclic AMP Signaling Pathway













Hs.416061
NM_005019
Phosphodiesterase
PDE1A
2q32.1
<0.01*
20.1




1A,




calmodulin-




dependent


Hs.9333
NM_173457
Phosphodiesterase
PDE8A
15q25.3
<0.01
16.3




8A


Hs.433700
NM_006823
cAMP-
PKIA
8q21.11
<0.05
14.2




dependent




Protein kinase




inhibitor alpha


Hs.183994
AK098311
Protein
PPP1CA
11q13
<0.01
13.1




phosphatase 1,




catalytic




subunit, alpha


Hs.303090
BX537399
Protein
PPP1R3C
10q23–q24






phosphatase 1,




regulatory




subunit 3C


Hs.166071
AK026533
Cyclin-
CDK5
7q36
<0.01
10.5




dependent







Phosphatidylinositol Signaling Pathway













Hs.374613
D26070
Inositol 1,4,5-
ITPR1
3p26–p25






triphosphate




receptor, type 1


Hs.460355
AL833252
Protein kinase
PRKCB1
16p11.2






C, beta 1


Hs.478199
NM_002740
Protein kinase
PRKCI
0
<0.01*
−29.5




C, iota


Hs.444924
NM_001263
CDP-
CDS1
4q21.23
<0.01*
−29.5




diacylglycerol


Hs.32309
AK093560
synthase 1
INPP1
2q32
<0.01*
21.5




polyphosphate-




1-phosphatase


Hs.369755
NM_014937
Inositol
INPP5F
10q26.11–q26.12






polyphosphate-




5-phosphatase F


Hs.528087
NM_002221
Inositol 1,4,5-
ITPKB
0
<0.01
14.8




trisphosphate 3-




kinase B


Hs.175343
BX648778
Phosphoinositide-
PIK3C2A
11p15.5–p14






3-kinase,




class 2, alpha




polypeptide


Hs.497487
Y11312
Phosphoinositide-
PIK3C2B
1q32
<0.01
16.6




3-kinase,




class 2, beta




polypeptide


Hs.132225
NM_181523
Phosphoinositide-
PIK3R1
5q13.1
<0.01*
−32.0




3-kinase,




regulatory




subunit 1


Hs.467192
AK090488
Protein
PPP2R1A
19q13.41
<0.01
12.1




phosphatase 2,




regulatory




subunit A, alpha




isoform


Hs.146339
NM_002717
Protein
PPP2R2A
8p21.2
<0.01*
32.3




phosphatase 2,




regulatory




subunit B, alpha




isoform














Major



Bipolar
Depressive



Disorder
Disorder












U133A
U95Av2
U133A
U133A



Cohort A
Cohort A
Cohort A
Cohort B















UniGene
P
% Fold
P
% Fold
P
% Fold
P
% Fold


ID
Value
Change
Value
Change
Value
Change
Value
Change










Monoamine Metabolism










Hs.370408
<0.01
11.5



Hs.46732
<0.01
18.9







Neuropeptide Ligand










Hs.1832
<0.01*
33.0



Hs.12409
<0.01*
22.1


Hs.1408
<0.01
−10.0







GPCRs















Hs.519057
<0.01*
24.4


<0.01*
21.9
<0.05
−17.7


Hs.131138


<0.01
−13.9
<0.01
−18.6
<0.01*
−24.7


Hs.112621
<0.01*
34.4


Hs.88372
<0.01*
−22.9


Hs.154210


<0.01
−19.7
<0.01*
−21.2
<0.01
−10.4


Hs.126667
<0.01
18.2


Hs.82002




<0.01*
−31.2
<0.01
−15.9


Hs.406094
<0.01*
38.3
<0.01*
−53.5
<0.01*
−40.5
<0.01*
−27.0


Hs.513633


<0.01
−12.0
<0.01
−15.9
<0.01
−14.9


Hs.148685
<0.01*
24.6
<0.01*
−36.5
<0.01*
−38.3
<0.01*
−30.3


Hs.99195


<0.05
−14.1
<0.05
−11.5







G protein and Regulators















Hs.134587
<0.01*
40.4








Hs.24950
<0.01*
−27.2


Hs.368733
<0.01
15.6
<0.01*
−28.2
<0.01*
−29.2
<0.01
−13.9







Cyclic AMP Signaling Pathway















Hs.416061
<0.01*
24.1








Hs.9333
<0.01
12.5
<0.01*
−24.3
<0.01*
−23.1
<0.01
−17.8


Hs.433700
<0.01
16.6


Hs.183994
<0.01
16.8


Hs.303090
<0.01
−15.4
<0.01*
−35.4
<0.01*
−63.2
<0.01*
−21.4


Hs.166071
<0.01
12.0







Phosphatidylinositol Signaling Pathway















Hs.374613


<0.05
13.0
<0.01
17.4




Hs.460355


<0.01
14.8
<0.01
11.5


Hs.478199


Hs.444924


Hs.32309
<0.01*
24.9


Hs.369755




<0.01*
21.8


Hs.528087
<0.05
11.5
<0.01
−12.7
<0.05
−12.3
<0.01*
−34.0


Hs.175343
<0.05
22.3
<0.05
−14.1
<0.01*
−35.9


Hs.497487
<0.01
14.8


Hs.132225


Hs.467192
<0.01
10.1


Hs.146339


















TABLE 14









Bipolar



Disorder



U95Av2



Cohort A













Name
Symbol
UniGene ID
GDB Acc #
G protein
Cytoband
P Value










Monoamine Metabolism













Catechol-O-
COMT
Hs.370408
AL390148

22q11.21–q11.23
<0.01


methyltransferase


Monoamine
MAOB
Hs.46732
NM_000898

Xp11.23
<0.01


oxidase B







Ligand peptide













Neuropeptide Y
NPY
Hs.1832
BF680552
Gi, Gq
7p15
<0.01*


Somatostatin
SST
Hs.12409
BI918626
Gi
3q28
<0.01*


Endothelin 3
EDN3
Hs.1408
BC053866
Gq
20q13.2–q13.3
<0.01







GPCRs













Neuropeptide Y
NPY1R
Hs.519057
L07615
Gi
4q31–q32



receptor Y1


Tachykinin
TACR2
Hs.88372
BC033742
Gq
10q11–q21



receptor 2


Neurotensin
NTSR2
Hs.131138
NM_012344
Gq
2p25.1



receptor 2


Endothelin
EDNRB
Hs.82002
NM_000115
Gq
13q22



receptor type B


Metabotropic
GRM3
Hs.112621
BC041407
Gi
7q21
<0.01*


Glutamate


receptor 3


Endothelial
EDG1
Hs.154210
BC018650
Gi, G12
1p21



differentiation


GPCR 1


Endothelial
EDG2
Hs.126667
BC036034
Gi, Gq,
9q31.3
<0.05


differentiation



G12


GPCR 2


G protein-coupled
GPR37
Hs.406094
BX649006
Unknown
7q31
<0.01*


receptor 37


G protein-coupled
GPRC5B
Hs.148685
NM_016235
Unknown
16p12
<0.01*


receptor C-5-B


G protein-coupled
GPR56
Hs.513633
NM_201524
Unknown
16q13



receptor 56


G protein-coupled
GPR125
Hs.99195
XM_291111
Unknown
4p15



receptor 125







Cyclic AMP Signaling Pathway













G protein alpha
GNAI1
Hs.134587
BC026326

7q21



inhibiting activity 1


Regulator of G-
RGS20
Hs.368733
AK094559

8q12



protein signalling


20


Phosphodiesterase
PDE1A
Hs.416061
NM_005019

2q32
<0.01*


1A


Phosphodiesterase
PDE8A
Hs.9333
NM_173457

15q25
<0.01


8A


Protein kinase A
PKIA
Hs.433700
NM_006823

8q21
<0.05


inhibitor alpha


Cyclin-dependent
CDK5
Hs.166071
AK026533

7q36
<0.01


kinase 5


Protein
PPP1CA
Hs.183994
AK098311

11q13
<0.01


phosphatase 1,


catalytic alpha


Protein
PPP1R3C
Hs.303090
BX537399

10q23–q24



phosphatase 1,


regulatory 3C







Phosphatidylinositol Signaling Pathway













Inositol
INPP5A
Hs.523360
NM_005539

10q26



polyphosphate-5-


phosphatase A


Inositol
INPP5F
Hs.369755
NM_014937

10q26



polyphosphate-5-


phosphatase F


Inositol 1,4,5-
ITPKB
Hs.528087
NM_002221

1q41–q43
<0.01


trisphosphate 3-


kinase B


Inositol
INPP1
Hs.32309
AK093560

2q32
<0.01*


polyphosphate-1-


phosphatase


CDP-
CDS1
Hs.444924
NM_001263

4q21.23
<0.01*


diacylglycerol


synthase 1


Phosphoinositide-
PIK3C2A
Hs.175343
BX648778

11p15–p14



3-kinase catalytic


2A


Phosphoinositide-
PIK3C2B
Hs.497487
Y11312

1q32
<0.01


3-kinase catalytic


2B


Phosphoinositide-
PIK3R1
Hs.132225
NM_181523

5q13
<0.01*


3-kinase


regulatory 1


Protein kinase C
PRKCI
Hs.478199
NM_002740

3p25–q27
<0.01*


iota


Inositol 1,4,5-
ITPR1
Hs.374613
D26070

3p26–p25



triphosphate


receptor 1


Protein kinase C
PRKCB1
Hs.460355
AL833252

16p11



beta 1













Bipolar




Disorder
Major Depressive Disorder













U95Av2
U133A
U95Av2
U133A
U133A



Cohort A
Cohort A
Cohort A
Cohort A
Cohort B
















Name
% FC
P Value
% FC
P Value
% FC
P Value
% FC
P Value
% FC










Monoamine Metabolism











Catechol-O-
15.8
<0.01
11.5



methyltransferase


Monoamine
15.9
<0.01
18.9


oxidase B







Ligand peptide











Neuropeptide Y
22.1
<0.01*
33.0



Somatostatin
29.0
<0.01*
22.1


Endothelin 3
−12.3
<0.01
−10.0







GPCRs
















Neuropeptide Y

<0.01*
24.4








receptor Y1


Tachykinin

<0.01*
−22.9


receptor 2


Neurotensin



<0.01
−13.9
<0.01
−18.6
<0.01*
−24.7


receptor 2


Endothelin





<0.01*
−31.2
<0.01
−15.9


receptor type B


Metabotropic
33.4
<0.01*
34.4


Glutamate


receptor 3


Endothelial



<0.01
−19.7
<0.01*
−21.2
<0.01
−10.4


differentiation


GPCR 1


Endothelial
24.1
<0.01
18.2


differentiation


GPCR 2


G protein-coupled
53.1
<0.01*
38.3
<0.01*
−53.5
<0.01*
−40.5
<0.01*
−27.0


receptor 37


G protein-coupled
38.4
<0.01*
24.6
<0.01*
−36.5
<0.01*
−38.3
<0.01*
−30.3


receptor C-5-B


G protein-coupled



<0.01
−12.0
<0.01
−15.9
<0.01
−14.9


receptor 56


G protein-coupled



<0.05
−14.1
<0.05
−11.5


receptor 125







Cyclic AMP Signaling Pathway
















G protein alpha

<0.01*
40.4








inhibiting activity 1


Regulator of G-



<0.01*
−28.2
<0.01*
−29.2
<0.01
−13.9


protein signalling


20


Phosphodiesterase
20.1
<0.01*
24.1


1A


Phosphodiesterase
16.3
<0.01
12.5
<0.01*
−24.3
<0.01*
−23.1
<0.01
−17.8


8A


Protein kinase A
14.2
<0.01
16.6


inhibitor alpha


Cyclin-dependent
10.5
<0.01
12.0


kinase 5


Protein
13.1
<0.01
16.8


phosphatase 1,


catalytic alpha


Protein



<0.01*
−35.4
<0.01*
−63.2
<0.01*
−21.4


phosphatase 1,


regulatory 3C







Phosphatidylinositol Signaling Pathway
















Inositol





<0.01
13.2
<0.05
12.2


polyphosphate-5-


phosphatase A


Inositol





<0.01*
21.8


polyphosphate-5-


phosphatase F


Inositol 1,4,5-
14.8
<0.05
11.5
<0.01
−12.7
<0.05
−12.3
<0.01*
−34.0


trisphosphate 3-


kinase B


Inositol
21.5
<0.01*
24.9


polyphosphate-1-


phosphatase


CDP-
−29.5


diacylglycerol


synthase 1


Phosphoinositide-



<0.05
−14.1
<0.01*
−35.9


3-kinase catalytic


2A


Phosphoinositide-
16.6
<0.01
14.8


3-kinase catalytic


2B


Phosphoinositide-
−32.0


3-kinase


regulatory 1


Protein kinase C
−29.5


iota


Inositol 1,4,5-



<0.05
13.0
<0.01
17.4


triphosphate


receptor 1


Protein kinase C



<0.01
14.8
<0.01
11.5


beta 1


















TABLE 15









Bipolar



Disorder



U95Av2



Cohort A
















UniGene
GDB


P



Name
Symbol
ID
Accession #
G protein
Cytoband
Value
% FC





Protein phosphatase
PPP1R3C
Hs.303090
BX537399

10q23–q24


1 regulatory 3C


Phosphodiesterase
PDE8A
Hs.9333
NM_173457

15q25


8A


Inositol 1,4,5-
ITPKB
Hs.528087
AJ242780

1q42


trisphosphate 3-


kinase B


G protein beta 5
GNB5
Hs.155090
AK092059

15q21


Somatostatin
SST
Hs.12409
BI918626
Gi
3q28
<0.01
−16.8


Adrenergic, beta-1-,
ADRB1
Hs.99913
BC045633
Gs
10q24–q26
<0.01
−18.4


receptor


Glutamate receptor,
GRM3
Hs.112621
BC041407
Gi
7q21
<0.01*
28.4


metabotropic 3


G protein-coupled
GPRC5B
Hs.148685
NM_016235
Unknown
16p12
<0.01*
31.8


receptor C-5-B


G protein-coupled
GPR37
Hs.406094
BX649006
Unknown
7q31


receptor 37


G protein-coupled
GPR56
Hs.513633
NM_201524
Unknown
16q13


receptor 56













Bipolar




Disorder
Major Depresive Disorder












U133A
U95Av2
U133A
U133A



Cohort A
Cohort A
Cohort A
Cohort B
















P

P

P

P



Name
Value
% FC
Value
% FC
Value
% FC
Value
% FC





Protein phosphatase


<0.01*
−26.4
<0.01*
−62.5
<0.01*
−29.6


1 regulatory 3C


Phosphodiesterase


<0.01*
−32.0


8A


Inositol 1,4,5-


<0.01
−12.5


<0.01*
−46.1


trisphosphate 3-


kinase B


G protein beta 5




<0.05
12.8
<0.01
18.7


Somatostatin
<0.01*
−25.7
<0.01*
−26.6
<0.01*
−34.2


Adrenergic, beta-1-,
<0.05
−13.4


receptor


Glutamate receptor,


metabotropic 3


G protein-coupled


<0.01*
−35.6
<0.01*
−34.3
<0.01*
−24.0


receptor C-5-B


G protein-coupled


<0.01*
−62.5


receptor 37


G protein-coupled


<0.01
−14.2
<0.01*
−20.0
<0.01*
−23.2


receptor 56



















TABLE 16









BPD
MDD



U95Av2
U95Av2
















Name
Symbol
UniGene ID
GDB Accession #
G protein
Cytoband
P Value
% FC
P Value
% FC



















Phosphodiesterase 4B
PDE4B
Hs.198072
CR749667

1p31


<0.01*
−45.3


Inositol polyphosphate-5-
INPP5A
Hs.523360
NM_005539

10q26
<0.01*
26.1


phosphatase A


Regulator of G-protein
RGS20
Hs.368733
AK094559

8q12


<0.01*
−23.8


signalling 20


Proenkephalin
PENK
Hs.339831
AK091563
Gi
8q23–q24
<0.01*
83.4
<0.01*
34.5


G protein-coupled receptor
GPRC5B
Hs.148685
NM_016235
Unknown
16p12


<0.01*
−31.7


C-5-B


G protein-coupled receptor
GPR37
Hs.406094
BX649006
Unknown
7q31


<0.01*
−42.4


37



















TABLE 17









BPD vs Control
MDD vs Control





















UniGene
Gene Name
Symbol
U95Av2
% FC
U133A
% FC
qRT-PCR
% FC
U95Av2
% FC
U133A
% FC
qRT-PCR
% FC
























Hs.1832
Neuropeptide
NPY
22.1
**
33.0
**
37.6
*









Y


Hs.12409
Somatostatin
SST
29.0
**
22.1
**
N.S.


Hs.148685
G protein-
GPRC5B
38.4
**
24.6
**
46.8
*
−36.5
**
−38.3
**
−54.0
**



coupled



receptor C-5-B


Hs.406094
G protein-
GPR37
53.1
**
38.3
**
54.1
*
−53.5
**
−40.5
**
−62.7
*



coupled



receptor 37


Hs.368733
Regulator of
RGS20






−28.2
**
−29.2
**
−36.9
**



G-protein



signalling 20


Hs.303090
Protein
PPP1R3C






−35.4
**
−63.2
**
−46.1
**



phosphatase 1



regulatory



subunit 3C


Hs.32309
Inositol
INPP1
21.5
**
24.9
**
21.7
*



polyphosphate-



1-phosphatase


















TABLE 18









BP - Control












UniGene
Amy 133A-22
HC 133A-22
nAcc 133A-22

















Name
Symbol
ID
UGRepAcc
Cytoband
p-value
% FC
p-value
% FC
p-value
% FC










Ligands

















Adrenomedullin
ADM
Hs.441047
CR603703
11p15
<0.01*
31.53






Brain-specific angiogenesis
BAI3
Hs.13261
AB011122
6q12
<0.01*
22.18
<0.01*
36.44


inhibitor 3


Cholecystokinin
CCK
Hs.458426
BC028133
3p22–p21


<0.01*
58.95
<0.01*
75.64


Somatostatin
SST
Hs.12409
BI918626
3q28
<0.01*
43.64


Chemokine (C—C motif)
CCL25
Hs.310511
CR603063
19p13


<0.01*
−23.50


ligand 25


Chemokine (C—X—C motif)
CXCL14
Hs.483444
NM_004887
5q31
<0.01*
−21.63
<0.01*
−37.40
<0.01*
−26.52


ligand 14


Frizzled homolog 7 (Drosophila)
FZD7
Hs.173859
AB017365
2q33


<0.01*
26.17


Monoamine oxidase B
MAOB
Hs.46732
NM_000898
Xp113
<0.01*
21.95


Neuropeptide Y
NPY
Hs.1832
BF680552
7p15
<0.01*
27.35


Neurotensin
NTS
Hs.80962
BF698911
12q21


<0.01*
31.10


Prodynorphin
PDYN
Hs.22584
BC026334
20pter–p12
<0.01*
73.04
<0.01*
20.40


Proenkephalin
PENK
Hs.339831
AK091563
8q23–q24
<0.01*
121.58


<0.01
19.08


GPCR


Neuropeptide Y receptor Y1
NPY1R
Hs.519057
L07615
4q31–q32
<0.01*
42.45


Gamma-aminobutyric acid
GABRD
Hs.113882
NM_000815
1p




<0.01*
27.99


(GABA) A receptor, delta


Neurotensin receptor 2
NTSR2
Hs.131138
NM_012344
2p25




<0.01*
−26.66


Oxytocin receptor
OXTR
Hs.2820
NM_000916
3p25




<0.01*
−27.98


Adenosine A2a receptor
ADORA2A
Hs.197029
BC013780
22q11
<0.01*
42.45


Cadherin, EGF LAG seven-pass
CELSR2
Hs.57652
AF234887
1p21


<0.01*
20.09


G-type receptor 2


G protein-coupled receptor 116
GPR116
Hs.362806
BC066121
6p12


<0.01*
−33.15


G protein-coupled receptor 125
GPR125
Hs.99195
XM_291111
4p15
<0.05
−11.08


G protein-coupled receptor 17
GPR17
Hs.46453
AK126849
2q21
<0.05
8.77


G protein-coupled receptor 22
GPR22
Hs.432557
AK122621
7q22–q31


<0.05
12.47


G protein-coupled receptor 37
GPR37
Hs.406094
BX649006
7q31


<0.01
−16.70


G protein-coupled receptor 51
GPR51
Hs.198612
AF056085
9q22–q22
<0.01
−19.60
<0.01
15.51
<0.01*
23.38


G protein-coupled receptor 6
GPR6
Hs.46332
NM_005284
6q21
<0.01*
78.20


<0.01*
24.14


G protein-coupled receptor,
GPRC5B
Hs.148685
NM_016235
16p12
<0.01
11.08


C-5-B


Glutamate receptor,
GRM3
Hs.112621
BC041407
7q21
<0.01*
28.68


metabotropic 3


Histamine receptor H3
HRH3
Hs.251399
NM_007232
20q13




<0.01
18.90


Dopamine receptor D1
DRD1
Hs.2624
NM_000794
5q35
<0.01*
53.37


<0.05
17.68


Endothelin receptor type B
EDNRB
Hs.82002
NM_000115
13q22


<0.01*
24.03
<0.01*
−37.40


5-hydroxytryptamine (serotonin)
HTR2C
Hs.149037
NM_000868
Xq24
<0.01*
97.85
<0.01*
24.17
<0.01*
33.87


receptor 2C


G protein


G protein, alpha inhibiting
GNAI1
Hs.134587
BC026326
7q21
<0.01*
22.98
<0.01*
32.60


activity polypeptide 1


Guanine nucleotide binding
GNB1
Hs.430425
AK123609
1p36


<0.01
19.19


protein, beta polypeptide 1


Guanine nucleotide binding
GNB5
Hs.155090
AK092059
15q21
<0.01
19.68
<0.01*
21.92


protein (G protein), beta 5


Guanine nucleotide binding
GNG3
Hs.179915
BM668891
11p11
<0.05
9.70
<0.01*
28.84
<0.01*
31.30


protein (G protein), gamma 3


Guanine nucleotide binding
GNG7
Hs.515544
AK024465
19p13
<0.01*
25.24


protein (G protein), gamma 7


Regulator of G protein signaling


Regulator of G-protein
RGS1
Hs.75256
AK093544
1q31


<0.05
−9.16


signalling 1


Regulator of G-protein
RGS2
Hs.78944
BC042755
1q31
<0.01*
23.56
<0.01*
20.60


signalling 2


Regulator of G-protein
RGS20
Hs.368733
AK094559
8q12


<0.01*
25.58


signalling 20


Regulator of G-protein
RGS4
Hs.386726
NM_005613
1q23


<0.05
−8.25
<0.01*
21.82


signalling 4


Regulator of G-protein
RGS5
Hs.24950
NM_003617
1q23
<0.01*
−26.85
<0.01*
−32.51
<0.01*
−20.65


signalling 5


Regulator of G-protein
RGS7
Hs.130171
CR627366
1q43
<0.01
18.39


signalling 7


Regulator of G-protein
RGS9
Hs.132327
BC022504
17q23–q24
<0.01*
25.58


signalling 9


Cyclic AMP signaling


Protein kinase,
PRKACB
Hs.487325
BX537705
1p36
<0.01*
26.80
<0.01*
20.88
<0.01*
22.95


cAMP-dependent,


catalytic, beta


Protein kinase,
PRKAR1A
Hs.280342
CR749311
17q23–q24
<0.01*
21.71
<0.01*
23.64
<0.01*
26.15


cAMP-dependent,


regulatory, type I, alpha


Protein kinase,
PRKAR2B
Hs.433068
BC075800
7q22
<0.01*
23.30
<0.01*
54.89


cAMP-dependent,


regulatory, type II, beta


Protein kinase
PKIA
Hs.433700
NM_006823
8q21


<0.01*
30.00


(cAMP-dependent,


catalytic) inhibitor alpha


Phosphodiesterase 8A
PDE8A
Hs.9333
NM_173457
15q25


<0.01*
−25.56


Cyclic AMP
ARPP-19
Hs.512908
AL833077
15q21
<0.01*
27.29


<0.01
19.35


phosphoprotein, 19 kD


Adenylate cyclase-associated
CAP2
Hs.132902
NM_006366
6p22
<0.05
10.52
<0.01*
35.43


protein, 2


Cyclin-dependent kinase 5
CDK5
Hs.166071
AK026533
7q36


<0.01*
20.85


Phosphatidylinositol signaling


Diacylglycerol kinase,
DGKB
Hs.487619
NM_004080
7p21
<0.01
18.61


<0.01*
26.46


beta 90 kDa


Inositol polyphosphate-5-
INPP5A
Hs.523360
NM_005539
10q26
<0.01*
34.39
<0.01*
27.86


phosphatase


Inositol polyphosphate-5-
INPP5F
Hs.369755
NM_014937
10q26
<0.01*
31.06
<0.01*
29.81


phosphatase F


Inositol 1,4,5-triphosphate
ITPR1
Hs.374613
D26070
3p26–p25
<0.01*
23.50
<0.01*
29.24


receptor, type 1


Phosphatidylinositol-4-
PIP5K1B
Hs.534371
BC030587
9q13
<0.01*
20.75


phosphate


5-kinase, type I, beta


Phosphatidylinositol-4-
PIP5K2C
Hs.144502
AK125526
12q13


<0.01*
23.03


phosphate


5-kinase, type II, gamma


Phospholipase C, beta 1
PLCB1
Hs.310537
NM_182734
20p12


<0.01
19.28


(phosphoinositide-


specific)


Protein kinase C, beta 1
PRKCB1
Hs.460355
AL833252
16p11
<0.01*
23.66
<0.01
18.25


Myristoylated alanine-rich
MARCKS
Hs.519909
NM_002356
6q22


<0.01*
23.12


protein kinase C substrate


Growth associated protein 43
GAP43
Hs.134974
AK091466
3q13


<0.01*
27.99
<0.01*
46.79


YWHA


Tyrosine 3-monooxygenase/
YWHAB
Hs.279920
NM_003404
20q13


<0.01*
37.20
<0.01*
22.45


tryptophan 5-monooxygenase


activation protein, beta


polypeptide


Chromosome 22 open reading
YWHAH
Hs.226755
CR622695
22q12


<0.01*
73.39
<0.01*
66.65


frame 24


Tyrosine 3-monooxygenase/
YWHAQ
Hs.74405
NM_006826
2p25


<0.01*
32.96


tryptophan 5-monooxygenase


activation protein, theta


polypeptide


Tyrosine 3-monooxygenase/
YWHAZ
Hs.492407
BC051814
8q23
<0.01
13.55
<0.01*
42.25
<0.01*
31.83


tryptophan 5-monooxygenase


activation protein, zeta


polypeptide


Calcium signaling


Calmodulin 1 (phosphorylase
CALM1
Hs.282410
BC047523
14q24–q31


<0.01*
21.80


kinase, delta)


Calmodulin 3 (phosphorylase
CALM3
Hs.515487
AK094964
19q13




<0.01*
30.65


kinase, delta)


Calcium/calmodulin-dependent
CAMK1
Hs.434875
AK094026
3p25


<0.05
18.27


protein kinase I


Calcium/calmodulin-dependent
CAMK2A
Hs.143535
NM_015981
5q32


<0.01*
20.41


protein kinase II alpha


Calcium/calmodulin-dependent
CAMK2B
Hs.351887
NM_001220
22q12


<0.01*
21.93


protein kinase II beta


Calmodulin binding transcription
CAMTA1
Hs.397705
NM_015215
1p36
<0.01
16.05
<0.05
14.94


activator 1


Doublecortin and CaM
DCAMKL1
Hs.507755
NM_004734
13q13




<0.01*
29.98


kinase-like 1


MAPK signaling


P21/Cdc42/Rac1-activated
PAK1
Hs.435714
NM_002576
11q13–q14
<0.05
13.71
<0.01
16.23


kinase 1 (STE20


homolog, yeast)


Mitogen-activated protein
MAP2K1
Hs.145442
NM_002755
15q22
<0.01*
33.40
<0.01*
57.55
<0.01*
46.39


kinase kinase 1


Mitogen-activated protein
MAP2K1IP1
Hs.433332
AK022313
4q23


<0.01*
20.94


kinase kinase 1


interacting protein 1


Mitogen-activated protein
MAP2K4
Hs.514681
AK131544
17p11
<0.01
17.47
<0.01*
29.05
<0.01*
22.97


kinase kinase 4


Mitogen-activated protein
MAP4K4
Hs.431550
NM_145686
2q11–q12
<0.01*
−23.32


kinase kinase kinase


kinase 4


Mitogen-activated
MAPK1
Hs.431850
NM_002745
22q11
<0.01*
31.80
<0.01*
52.27
<0.01*
41.35


protein kinase 1


Mitogen-activated
MAPK10
Hs.25209
AK124791
4q22–q23


<0.01*
25.47


protein kinase 10


Mitogen-activated
MAPK9
Hs.484371
BC032539
5q35


<0.01
13.93
<0.01*
22.88


protein kinase 9


Protein Phosphatase


Protein phosphatase 1,
PPP1CC
Hs.79081
NM_002710
12q24


<0.01*
25.65


catalytic subunit,


gamma isoform


Protein phosphatase 1,
PPP1R2
Hs.184840
NM_006241
3q29


<0.01*
21.53


regulatory (inhibitor)


subunit 2


Protein phosphatase 1,
PPP1R3C
Hs.303090
BX537399
10q23–q24
<0.01
−16.80


<0.01*
−47.55


regulatory subunit 3C


Protein phosphatase 2C,
PPM2C
Hs.22265
NM_018444
8q22
<0.05
23.33


magnesium-


dependent, catalytic subunit


Protein phosphatase 2 (formerly
PPP2CA
Hs.483408
BX640662
5q23–q31


<0.01*
24.59


2A), catalytic


subunit, alpha isoform


Protein phosphatase 2 (formerly
PPP2R1A
Hs.467192
AK090488
19q13


<0.01*
27.34
<0.01*
24.11


2A), regulatory subunit A


(PR 65), alpha isoform


Protein phosphatase 2 (formerly
PPP2R2B
Hs.193825
M64930
5q31–5q32


<0.01*
38.94


2A), regulatory subunit B


(PR 52), beta isofrom


Protein phosphatase 2,
PPP2R5C
Hs.368264
NM_002719
14q32


<0.01*
23.54


regulatory subunit B


(B56), gamma isoform


Protein phosphatase 3
PPP3CA
Hs.435512
NM_000944
4q21–q24
<0.01*
29.94
<0.01*
23.23


(formerly 2B), catalytic


subunit, alpha isoform


(calcineurin A alpha)


Protein phosphatase 3
PPP3CB
Hs.500067
BC028049
10q21–q22
<0.01*
20.95
<0.01*
45.82
<0.01*
31.17


(formerly 2B), catalytic


subunit, beta isform


(calcineurin A beta)


Protein phosphatase 3
PPP3R1
Hs.280604
BC027913
2p15


<0.01*
25.69


(formerly 2B), regulatory


subunit B, 19 kDa, alpha


isoform (calcineurin B,


type I)


AP1


V-fos FBJ murine osteosarcoma
FOS
Hs.25647
BX647104
14q24


<0.01*
−40.86
<0.01*
−80.23


viral oncogene homolog


V-jun sarcoma virus 17
JUN
Hs.525704
NM_002228
1p32–p31
<0.01*
−25.24


oncogene homolog (avian)


Small G protein


Rho guanine nucleotide
ARHGEF3
Hs.476402
AL833224
3p21–p13


<0.01*
31.85


exchange factor (GEF) 3


Ras homolog gene family,
ARHI
Hs.194695
AK096393
1p31
<0.01*
132.94


<0.01*
33.15


member I


DIRAS family, GTP-binding
DIRAS2
Hs.165636
NM_017594
9q22


RAS-like 2


RAB31, member RAS
RAB31
Hs.99528
NM_006868
18p11
<0.05
−9.44


oncogene family


RAB33A, member
RAB33A
Hs.56294
AK094927
Xq25




<0.01*
21.72


RAS oncogene family


Rab acceptor 1 (prenylated)
RABAC1
Hs.11417
BE779053
19q13




<0.01*
21.97


RAN binding protein 6
RANBP6
Hs.167496
BX537405
9p24


<0.01*
25.82


RAP1, GTPase
RAP1GA1
Hs.148178
BC035030
1p36–p35
<0.01*
52.74


activating protein 1


Hypothetical protein
RASGRF1
Hs.459035
NM_002891
15q24


<0.01*
28.36


LOC145899


RAS guanyl releasing protein 1
RASGRP1
Hs.511010
AF081195
15q15
<0.01*
24.37


(calcium and DAG-regulated)


RAS guanyl releasing protein 3
RASGRP3
Hs.143674
BC027849
2p25–p24


<0.01
−14.27


(calcium and DAG-regulated)


Ras-like without CAAX 2
RIT2
Hs.464985
AL713637
18q12




<0.01*
32.56


RAS-related on chromosome 22
RRP22
Hs.73088
NM_001007279
22q12




<0.01*
20.87


V-Ki-ras2 Kirsten rat sarcoma 2
KRAS2
Hs.505033
NM_033360
12p12


<0.01*
22.38


viral oncogene homolog


Ras-GTPase activating protein
G3BP2
Hs.303676
NM_203505
4q21


<0.01*
23.35
<0.01*
36.80


SH3 domain-


binding protein 2


Potassium channel


Potassium channel
KCMF1
Hs.345694
NM_020122
2p11


<0.01*
22.07


modulatory factor 1


Potassium voltage-gated
KCNAB2
Hs.440497
AK124696
1p36


<0.01*
24.09


channel, shaker-


related beta 2


Potassium voltage-gated
KCND2
Hs.21703
AB028967
7q31


<0.01*
20.05


channel, Shal-related 2


Potassium inwardly-rectifying
KCNJ2
Hs.1547
NM_000891
17q23–q24


<0.01*
−28.13


channel, subfamily J 2


Potassium inwardly-rectifying
KCNJ6
Hs.50927
AK058042
21q22


<0.01*
26.12


channel, subfamily J 6


Potassium inwardly-rectifying
KCNJ13
Hs.467338
NM_002242
2q37


<0.01*
31.87


channel, subfamily J 13


Potassium channel, subfamily
KCNK1
Hs.208544
AL833343
1q42–q43




<0.01*
23.33


K, member 1


Potassium intermediate/
KCNN2
Hs.98280
NM_021614
5q22


<0.01*
28.96


small conductance


calcium-activated


channel, subfamily N,


member 2


Potassium intermediate/
KCNN3
Hs.490765
BX649146
1q21
<0.01
−13.90


small conductance


calcium-activated


channel, subfamily N,


member 3


Potassium channel,
KCNV1
Hs.13285
NM_014379
8q22–q24


<0.01
11.16


subfamily V, member 1


Sodium channel


Sodium channel,
SCN3B
Hs.4865
AB032984
11q24


<0.01*
41.71
<0.01*
37.41


voltage-gated, type III, beta


Solute carrier family 12,
SLC12A5
Hs.21413
NM_020708
20q132
<0.01
11.53
<0.01*
21.22


(potassium-chloride


transporter) member 5


Solute carrier family 24
SLC24A3
Hs.211252
AL833544
20p13


<0.01*
60.32


(sodium/potassium/


calcium exchanger),


member 3


Voltage-dependent
VDAC1
Hs.519320
AK122953
5q31


<0.01*
27.48


anion channel 1


Calcium Channel


Calcium channel,
CACNA2D3
Hs.369421
NM_018398
3p21


<0.01*
36.34


voltage-dependent, alpha


2/delta 3


Calcium channel,
CACNB2
Hs.59093
NM_000724
10p12


<0.01*
21.04
<0.01*
23.85


voltage-dependent, beta 2


subunit


Calcium channel, 
CACNB3
Hs.250712
AK122911
12q13


<0.01*
22.11


voltage-dependent, beta 3


subunit


Calcium channel, 
CACNG3
Hs.7235
AK095553
16p12–p13


<0.01*
32.54


voltage-dependent, gamma


subunit 3


Chloride channel


Chloride intracellular channel 4
CLIC4
Hs.440544
AL117424
1p36


<0.05
−17.97


















TABLE 19









MD - Control












UniGene
Amy 133A-22
HC 133A-22
nAcc 133A-22

















Name
Symbol
ID
UGRepAcc
Cytoband
p-value
% FC
p-value
% FC
p-value
% FC










Ligands

















Adrenomedullin
ADM
Hs.441047
CR603703
11p15
<0.05
−7.16
<0.01*
−34.24




Brain-specific
BAI3
Hs.13261
AB011122
6q12
<0.01*
20.13
<0.01*
20.61


angiogenesis inhibitor 3


Cholecystokinin
CCK
Hs.458426
BC028133
3p22–p21
<0.01*
29.74
<0.01*
81.09


Somatostatin
SST
Hs.12409
BI918626
3q28
<0.01*
20.70


Frizzled homolog 7
FZD7
Hs.173859
AB017365
2q33


<0.05
8.11


(Drosophila)


Latrophilin 2
LPHN2
Hs.24212
AF104266
1p31
<0.01*
29.33


Prodynorphin
PDYN
Hs.22584
BC026334
20pter–p12
<0.01*
34.43


<0.01*
31.55


Proenkephalin
PENK
Hs.339831
AK091563
8q23–q24
<0.01*
57.92
<0.01*
21.80
<0.01*
22.13


Prostaglandin D2 synthase
PTGDS
Hs.446429
BM805807
9q34–q34


<0.01*
−23.24


21 kDa (brain)


GPCR


Neuropeptide Y receptor Y1
NPY1R
Hs.519057
L07615
4q31–q32
<0.01*
44.09


Gamma-aminobutyric
GABRD
Hs.113882
NM_000815
1p


<0.01*
22.04
<0.01*
22.85


acid (GABA) A


receptor, delta


Neurotensin receptor 2
NTSR2
Hs.131138
NM_012344
2p25
<0.01*
−37.48


<0.01
−17.42


Oxytocin receptor
OXTR
Hs.2820
NM_000916
3p25




<0.01
−15.90


Cholecystokinin B receptor
CCKBR
Hs.203
AF239668
11p15


<0.01*
27.01


Adenosine A2a receptor
ADORA2A
Hs.197029
BC013780
22q11
<0.01
18.30


<0.01*
23.23


Angiotensin II receptor-like 1
AGTRL1
Hs.438311
AK075252
11q12


<0.01*
−22.99


G protein-coupled receptor 125
GPR125
Hs.99195
XM_291111
4p15
<0.01*
−22.88


G protein-coupled receptor 17
GPR17
Hs.46453
AK126849
2q21
<0.01*
−27.08


G protein-coupled receptor 22
GPR22
Hs.432557
AK122621
7q22–q31


<0.01*
24.51


G protein-coupled receptor 37
GPR37
Hs.406094
BX649006
7q31


<0.01*
−43.90


G-protein coupled receptor
GPR37L1
Hs.132049
BC050334
1q32
<0.01*
−27.67


37 like 1


G protein-coupled receptor 51
GPR51
Hs.198612
AF056085
9q22–q22
<0.01*
31.80
<0.01*
29.07


G protein-coupled receptor 56
GPR56
Hs.513633
NM_201524
16q13
<0.01*
−24.98


G protein-coupled receptor 6
GPR6
Hs.46332
NM_005284
6q21
<0.01*
27.88


<0.01*
41.21


Chemokine (C—X—C motif)
CXCR4
Hs.421986
CR614663
2q21


<0.01*
−20.68


receptor 4


G protein-coupled
GPRC5B
Hs.148685
NM_016235
16p12
<0.01*
−27.94
<0.01*
−36.01


receptor, C-5-B


Histamine receptor H3
HRH3
Hs.251399
NM_007232
20q13




<0.01*
30.42


Dopamine receptor D1
DRD1
Hs.2624
NM_000794
5q35
<0.01*
27.79


<0.01*
25.88


Endothelial differentiation,
EDG1
Hs.154210
BC018650
1p21
<0.01*
−20.04


sphingolipid


GPCR 1


Endothelin receptor
EDNRB
Hs.82002
NM_000115
13q22
<0.01*
−64.83
<0.01
−14.45
<0.01*
−37.17


type B


5-hydroxytryptamine
HTR2A
Hs.424980
NM_000621
13q14–q21
<0.01*
45.03
<0.01*
42.32


(serotonin) receptor 2A


5-hydroxytryptamine
HTR2C
Hs.149037
NM_000868
Xq24
<0.01
14.27
<0.01
−18.49
<0.01*
31.98


(serotonin) receptor 2C


G protein


Guanine nucleotide
GNA13
Hs.515018
NM_006572
17q24
<0.01*
−20.84


binding protein (G


protein), alpha 13


G protein, alpha
GNAI1
Hs.134587
BC026326
7q21
<0.05
17.13


inhibiting activity


polypeptide 1


Guanine nucleotide
GNB1
Hs.430425
AK123609
1p36


<0.01*
22.06


binding protein, beta


polypeptide 1


Guanine nucleotide
GNB5
Hs.155090
AK092059
15q21
<0.01*
24.99
<0.01*
32.65


binding protein (G


protein), beta 5


Guanine nucleotide
GNG12
Hs.431101
NM_018841
1p31
<0.01*
−21.46


binding protein (G


protein), gamma 12


Guanine nucleotide
GNG3
Hs.179915
BM668891
11p11
<0.01*
26.57
<0.01*
41.14
<0.05
14.55


binding protein (G


protein), gamma 3


Regulator of G protein signaling


Regulator of G-protein signalling 1
RGS1
Hs.75256
AK093544
1q31


<0.01*
−34.56


Regulator of G-protein signalling 2
RGS2
Hs.78944
BC042755
1q31
<0.01*
36.07
<0.01
17.15


Regulator of G-protein
RGS20
Hs.368733
AK094559
8q12
<0.01*
−32.34


signalling 20


Regulator of G-protein signalling 4
RGS4
Hs.386726
NM_005613
1q23
<0.01*
72.22
<0.01*
40.31
<0.05
10.50


Regulator of G-protein signalling 5
RGS5
Hs.24950
NM_003617
1q23
<0.01
8.03


Regulator of G-protein signalling 7
RGS7
Hs.130171
CR627366
1q43
<0.01*
36.06
<0.01*
36.42


Regulator of G-protein signalling 9
RGS9
Hs.132327
BC022504
17q23–q24
<0.01
11.68


Cyclic AMP signaling


Protein kinase, cAMP-
PRKACB
Hs.487325
BX537705
1p36
<0.01
17.60
<0.01
16.51


dependent, catalytic,


beta


Protein kinase,
PRKAR1A
Hs.280342
CR749311
17q23–q24
<0.01
11.92
<0.05
10.27


cAMP-dependent,


regulatory, type I, alpha


(tissue specific


extinguisher 1)


Protein kinase,
PRKAR2B
Hs.433068
BC075800
7q22
<0.01*
26.41
<0.01*
52.17


cAMP-dependent,


regulatory,


type II, beta


Protein kinase (cAMP-
PKIA
Hs.433700
NM_006823
8q21


<0.05
14.78


dependent, catalytic)


inhibitor alpha


Phosphodiesterase
PDE4DIP
Hs.487925
NM_014644
1q12
<0.01*
−21.98


4D interacting protein


(myomegalin)


Phosphodiesterase 8A
PDE8A
Hs.9333
NM_173457
15q25
<0.01*
−20.78
<0.01*
−39.39


Phosphodiesterase 8B
PDE8B
Hs.78106
AF079529
5q13
<0.01*
20.36


Cyclic AMP phosphoprotein,
ARPP-19
Hs.512908
AL833077
15q21
<0.01*
24.18
<0.01*
25.33
<0.01*
27.45


19 kD


Adenylate cyclase-
CAP2
Hs.132902
NM_006366
6p22
<0.01*
32.38
<0.01*
27.21


associated protein, 2


Cyclin-dependent
CDK5
Hs.166071
AK026533
7q36


<0.01
18.67


kinase 5


Phosphatidylinositol signaling


Diacylglycerol kinase,
DGKB
Hs.487619
NM_004080
7p21
<0.01*
32.70


<0.01*
26.36


beta 90 kDa


Inositol polyphosphate-
INPP5A
Hs.523360
NM_005539
10q26
<0.01*
22.52
<0.01*
40.16
<0.01*
22.98


5-phosphatase, 40 kDa


Inositol polyphosphate-
INPP5F
Hs.369755
NM_014937
10q26
<0.01*
40.58
<0.01*
45.18
<0.01*
32.42


5-phosphatase F


Inositol 1,4,5-trisphosphate
ITPKA
Hs.2722
BC026331
15q14–q21
<0.01*
29.49
<0.01*
32.18


3-kinase A


Inositol 1,4,5-trisphosphate
ITPKB
Hs.528087
AJ242780
1q423
<0.01*
−34.51
<0.01*
−34.14


3-kinase B


Inositol 1,4,5-triphosphate
ITPR1
Hs.374613
D26070
3p26–p25
<0.01*
45.62
<0.01*
51.25


receptor, type 1


Phosphoinositide-3-kinase,
PIK3C2A
Hs.175343
BX648778
11p15–p14
<0.01*
−63.06


class 2, alpha


polypeptide


Phosphoinositide-3-kinase,
PIK3C2A
Hs.175343
BX648778
11p15–p14
<0.01*
−63.06


class 2, alpha


polypeptide


Phosphatidylinositol-4-
PIP5K1B
Hs.534371
BC030587
9q13
<0.01*
24.44


phosphate 5-kinase,


type I, beta


Phosphatidylinositol-4-
PIP5K2C
Hs.144502
AK125526
12q13


<0.05
17.13


phosphate 5-kinase,


type II, gamma


Phospholipase C, beta
PLCB1
Hs.310537
NM_182734
20p12


<0.01*
26.19


1 (phosphoinositide-


specific)


Protein kinase C, beta 1
PRKCB1
Hs.460355
AL833252
16p11
<0.01*
51.20
<0.01*
43.94


Growth associated
GAP43
Hs.134974
AK091466
3q13


<0.01*
49.55
<0.01*
30.12


protein 43


YWHA


Tyrosine 3-monooxygenase/
YWHAB
Hs.279920
NM_003404
20q13
<0.01*
20.42
<0.01*
30.24
<0.05
13.58


tryptophan 5-


monooxygenase


activation protein, beta


polypeptide


Chromosome 22 open
YWHAH
Hs.226755
CR622695
22q12
<0.01*
31.18
<0.01*
56.74
<0.01*
26.27


reading frame 24


Tyrosine 3-monooxygenase/
YWHAZ
Hs.492407
BC051814
8q23
<0.01*
20.06
<0.01*
30.03
<0.01*
20.91


tryptophan 5-


monooxygenase


activation protein, zeta


polypeptide


Calcium signaling


Calmodulin 1 (phosphorylase
CALM1
Hs.282410
BC047523
14q24–q31


<0.01
16.48


kinase, delta)


Calmodulin 3 (phosphorylase
CALM3
Hs.515487
AK094964
19q13




<0.01
14.08


kinase, delta)


Calcium/calmodulin-
CAMK1
Hs.434875
AK094026
3p25


<0.01*
20.75


dependent protein


kinase I


Calcium/calmodulin-
CAMK2A
Hs.143535
NM_015981
5q32


<0.01*
30.59


dependent protein


kinase II alpha


Calcium/calmodulin-
CAMK2B
Hs.351887
NM_001220
22q12


<0.05
13.91


dependent protein


kinase II beta


Calcium/calmodulin-
CaMKIINalpha
Hs.197922
CR604926
1p36


<0.01*
21.16


dependent protein


kinase II


Calcium/calmodulin-
CAMKK2
Hs.297343
NM_006549
12q24


<0.01*
22.16


dependent protein


kinase kinase 2, beta


Calmodulin binding 
CAMTA1
Hs.397705
NM_015215
1p36
<0.01*
31.29
<0.01*
41.27


transcription activator 1


Doublecortin and CaM 
DCAMKL1
Hs.507755
NM_004734
13q13




<0.05
16.77


kinase-like 1


MAPK signaling


P21/Cdc42/Rac1-
PAK1
Hs.435714
NM_002576
11q13–q14
<0.01*
32.71
<0.01*
36.58


activated kinase 1


(STE20 homolog, yeast)


Mitogen-activated 
MAP2K1
Hs.145442
NM_002755
15q22
<0.01*
23.61
<0.01*
35.94
<0.01*
26.19


protein kinase kinase 1


Mitogen-activated 
MAP2K1IP1
Hs.433332
AK022313
4q23


<0.05
13.15


protein kinase kinase 1


interacting protein 1


Mitogen-activated 
MAP2K4
Hs.514681
AK131544
17p11
<0.01*
23.15
<0.01
19.77
<0.05
8.77


protein kinase kinase 4


Mitogen-activated 
MAP4K4
Hs.431550
NM_145686
2q11–q12
<0.01*
−24.08


protein kinase kinase


kinase kinase 4


Mitogen-activated 
MAP4K5
Hs.130491
NM_198794
14q11–q21


<0.01*
−24.23


protein kinase kinase


kinase kinase 5


Mitogen-activated 
MAPK1
Hs.431850
NM_002745
22q11
<0.05
9.57
<0.01*
22.13


protein kinase 1


Mitogen-activated 
MAPK10
Hs.25209
AK124791
4q22–q23


<0.01*
21.09


protein kinase 10


Mitogen-activated 
MAPK6
Hs.411847
NM_002748
15q21


<0.01*
23.03


protein kinase 6


Mitogen-activated 
MAPK9
Hs.484371
BC032539
5q35


<0.01*
21.30
<0.05
12.07


protein kinase 9


Protein Phosphatase


Protein phosphatase 
PPP1R2
Hs.184840
NM_006241
3q29


<0.01*
20.02


1, regulatory


(inhibitor) subunit 2


Protein phosphatase 
PPP1R3C
Hs.303090
BX537399
10q23–q24
<0.01*
−54.15
<0.01*
−47.47
<0.01*
−43.72


1, regulatory subunit


3C


Protein phosphatase 2C, 
PPM2C
Hs.22265
NM_018444
8q22
<0.01*
35.09
<0.01*
47.45


magnesium-


dependent, catalytic subunit


Protein phosphatase 
PPP2CA
Hs.483408
BX640662
5q23–q31


<0.01
17.92


2 (formerly 2A),


catalytic subunit,


alpha isoform


Protein phosphatase 2 
PPP2R1A
Hs.467192
AK090488
19q13


<0.05
16.04
<0.05
12.64


(formerly 2A),


regulatory subunit A (PR 65),


alpha isoform


Protein phosphatase 
PPP2R2B
Hs.193825
M64930
5q31–5q32


<0.01*
29.87


2 (formerly 2A),


regulatory subunit B


(PR 52), beta isoform


Protein phosphatase 2, 
PPP2R5C
Hs.368264
NM_002719
14q32


<0.01
18.07


regulatory subunit


B (B56), gamma isoform


Protein phosphatase
PPP3CA
Hs.435512
NM_000944
4q21–q24
<0.01*
43.20
<0.01*
26.06


3 (formerly 2B),


catalytic subunit,


alpha isoform (calcineurin


A alpha)


Protein phosphatase 
PPP3CB
Hs.500067
BC028049
10q21–q22
<0.01*
37.18
<0.01*
47.07
<0.01
17.08


3 (formerly 2B),


catalytic subunit,


beta isoform (calcineurin


A beta)


Protein phosphatase 
PPP3R1
Hs.280604
BC027913
2p15
<0.01*
33.47
<0.01*
31.25


3 (formerly 2B),


regulatory subunit B,


19 kDa, alpha isoform


(calcineurin B, type I)


AP1


V-fos FBJ murine 
FOS
Hs.25647
BX647104
14q24


<0.01*
−55.69
<0.01*
−46.49


osteosarcoma viral


oncogene homolog


Small G protein


Rho guanine nucleotide 
ARHGEF3
Hs.476402
AL833224
3p21–p13


<0.01*
28.51


exchange factor


(GEF) 3


Ras homolog gene 
ARHI
Hs.194695
AK096393
1p31
<0.05
20.60


<0.01*
28.06


family, member I


DIRAS family, 
DIRAS2
Hs.165636
NM_017594
9q22
<0.01*
63.78


GTP-binding RAS-like 2


RAB31, member RAS
RAB31
Hs.99528
NM_006868
18p11
<0.01*
−27.47


oncogene family


Rab acceptor 1 (prenylated)
RABAC1
Hs.11417
BE779053
19q13




<0.01
16.11


RAN binding protein 6
RANBP6
Hs.167496
BX537405
9p24


<0.05
17.28


RAP1, GTPase 
RAP1GA1
Hs.148178
BC035030
1p36–p35
<0.01*
22.75


activating protein 1


Hypothetical protein 
RASGRF1
Hs.459035
NM_002891
15q24


<0.01*
26.84


LOC145899


RAS guanyl releasing
RASGRP1
Hs.511010
AF081195
15q15
<0.05
18.52


protein 1 (calcium


and DAG-regulated)


RAS guanyl releasing
RASGRP3
Hs.143674
BC027849
2p25–p24


<0.01*
−20.95


protein 3 (calcium


and DAG-regulated)


Rho-related BTB 
RHOBTB3
Hs.445030
NM_014899
5q15
<0.01*
−24.67


domain containing 3


Ras and Rab interactor 2
RIN2
Hs.472270
NM_018993



<0.01*
−37.60


Ras-like without CAAX 2
RIT2
Hs.464985
AL713637
18q12




<0.01*
29.34


Rho family GTPase 1
RND1
Hs.124940
AK124288
12q12–q13


<0.01*
28.02


V-Ki-ras2 Kirsten 
KRAS2
Hs.505033
NM_033360
12p12


<0.01*
20.01


rat sarcoma 2 viral


oncogene homolog


Ras-GTPase activating 
G3BP2
Hs.303676
NM_203505
4q21


<0.01*
25.13
<0.05
17.74


protein SH3


domain-binding protein 2


Potassium channel


Potassium channel
KCMF1
Hs.345694
NM_020122
2p11


<0.01*
23.55


modulatory factor 1


Potassium voltage-
KCNAB2
Hs.440497
AK124696
1p36


<0.05
19.97


gated channel, shaker-


related beta 2


Potassium inwardly-
KCNJ2
Hs.1547
NM_000891
17q23–q24


<0.01*
−41.14


rectifying channel,


subfamily J 2


Potassium inwardly-
KCNJ10
Hs.408960
NM_002241
1q22–q23




<0.01*
−25.25


rectifying channel,


subfamily J 10


Potassium channel, 
KCNK1
Hs.208544
AL833343
1q42–q43




<0.05
11.05


subfamily K, member 1


Potassium intermediate/
KCNN3
Hs.490765
BX649146
1q21
<0.01*
−30.93
<0.01*
−23.10


small conductance


calcium-activated


channel, subfamily N,


member 3


Potassium channel, 
KCNV1
Hs.13285
NM_014379
8q22–q24


<0.01*
21.10


subfamily V, member 1


Sodium channel


Sodium channel, 
SCN2A2
Hs.470470
NM_021007
2q23–q24


<0.01*
26.22


voltage-gated, type II,


alpha 2


Sodium channel, 
SCN3B
Hs.4865
AB032984
11q24


<0.01*
22.62
<0.01
17.61


voltage-gated, type III,


beta


Solute carrier family 
SLC12A5
Hs.21413
NM_020708
20q132
<0.01*
26.35
<0.01*
23.76


12, (potassium-


chloride transporter)


member 5


Voltage-dependent 
VDAC1
Hs.519320
AK122953
5q31


<0.01
18.44


anion channel 1


Calcium Channel


Calcium channel, 
CACNA2D3
Hs.369421
NM_018398
3p21


<0.01*
31.61


voltage-dependent, alpha


2/delta 3


Calcium channel,
CACNB2
Hs.59093
NM_000724
10p12


<0.01*
32.21


voltage-dependent, beta


2 subunit


Calcium channel, 
CACNB3
Hs.250712
AK122911
12q13


<0.01*
43.88


voltage-dependent, beta


3 subunit


Calcium channel, 
CACNG3
Hs.7235
AK095553
16p12–p13


<0.01*
32.10


voltage-dependent,


gamma subunit 3


Chloride channel


Chloride intracellular
CLIC4
Hs.440544
AL117424
1p36


<0.01*
−34.30


channel 4



















TABLE 20










Dorsalateral



Anterior Cingulate Cortex
Prefrontal Cortex



























Chromosomal
Unigene

FC
FC
FC


T
FC
FC
FC


T


Gene Symbol
Accession
Gene Name
Location
clusters
Pathway
BPD
MDD
Control
T BPD
T MDD
Control
BPD
MDD
Control
T BPD
T MDD
Control



























HSPA2
NM_021979
heat shock 70 kDa
14q24.1
Hs.432648
Chaperone
1.36
0.68
0.69
9.00
−11.58
−12.22
1.21
0.69
1.01
5.95
−12.25
0.36




protein 2


SPP1
NM_000582
secreted
4q21–q25
Hs.313
Apoptosis
1.28
0.84
0.36
7.67
−5.88
−35.42
1.32
0.86
0.70
8.49
−5.10
−12.35




phosphoprotein 1




(osteopontin, bone




sialoprotein I, early




T-lymphocyte




activation 1)


TM4SF10
NM_031442
transmembrane 4
Xp11.4
Hs.8769
Apoptosis
1.27
0.86
0.75
7.34
−4.73
−10.03
1.48
0.92
0.69
10.52
−2.30
−10.96




superfamily




member 10


CAT
NM_001752
catalase
11p13
Hs.395771
Oxidative
1.22
0.89
0.68
6.28
−3.94
−13.28
1.22
0.85
0.78
6.14
−4.94
−8.30







Stress


S100B
NM_006272
S100 calcium
21q22.3
Hs.422181
Apoptosis
1.21
0.94
0.68
6.39
−2.25
−14.39
1.19
0.89
0.75
5.16
−3.57
−9.70




binding protein,




beta (neural)


NR4A1
NM_173157
nuclear receptor
12q13
Hs.1119
Mitochondria
0.83
0.86
1.17
−9.63
−8.30
9.19
0.83
0.90
1.15
8.36
−5.21
7.06




subfamily 4, group




A, member 1


BZRAP1
NM_004758
benzodiazapine
17q22–q23
Hs.112499
Mitochondria
0.82
1.02
1.16
−7.05
0.68
6.01
1.00
0.96
1.21
0.10
−1.13
5.47




receptor




(peripheral)




associated protein 1


GSK3B
NM_002093
glycogen synthase
3q13.3
Hs.282359
Apoptosis
0.76
1.03
1.20
−9.16
0.94
7.11
0.93
1.00
1.05
−2.43
0.02
1.76




kinase 3 beta


COX7A1
NM_001864
cytochrome c
19q13.1
Hs.421621
Mitochondria
0.73
1.15
1.36
−7.65
3.54
8.57
0.94
1.08
1.16
−1.73
2.08
4.48




oxidase subunit VIIa




polypeptide 1




(muscle)


UQCRB
NM_006294
ubiquinol-
8q22
Hs.131255
Mitochondria
1.06
1.20
0.98
1.64
5.37
−0.58
1.15
1.09
1.14
3.48
2.16
3.62




cytochrome c




reductase binding




protein


DUSP1
NM_004417
dual specificity
5q34
Hs.171695
Oxidative
0.87
0.74
1.10
−4.00
−8.85
2.92
0.84
0.83
1.13
−4.06
−4.54
3.31




phosphatase 1


Stress


DUSP6
NM_001946
dual specificity
12q22–q23
Hs.298654
Apoptosis
0.84
0.70
1.59
−4.15
−8.89
12.26
0.96
0.77
1.77
−0.93
−5.46
13.19




phosphatase 6


TM4SF10
NM_031442
transmembrane 4
Xp11.4
Hs.8769
Apoptosis
1.27
0.86
0.75
7.34
−4.73
−10.03
1.48
0.92
0.69
10.52
−2.30
−10.96




superfamily




member 10


ATP6V0E
NM_003945
ATPase, H+
5q35.2
Hs.440165
Lysosyme
1.15
0.88
0.59
2.89
−2.87
−12.33
1.36
0.90
0.63
6.10
−2.23
−10.14




transporting,




lysosomal 9 kDa, V0




subunit e


GLUL
S70290
glutamate-ammonia
1q31
Hs.442669
Mitochondria
1.00
0.67
0.78
−0.07
−7.65
−5.22
1.33
0.78
0.70
4.96
−4.42
−7.06




ligase (glutamine




synthase)


SPP1
NM_000582
secreted
4q21–q25
Hs.313
Apoptosis
1.28
0.84
0.36
7.67
−5.88
−35.42
1.32
0.86
0.70
8.49
−5.10
−12.35




phosphoprotein 1




(osteopontin, bone




sialoprotein I, early




T-lymphocyte




activation 1)


APG-1
NM_014278
heat shock protein
4q28
Hs.135554
Chaperone
0.98
1.05
1.06
−0.22
0.64
0.92
1.26
1.19
0.95
2.81
2.20
−0.77




(hsp110 family)


HSPA5
NM_005347
heat shock 70 kDa
9q33–q34.1
Hs.310769
Chaperone
1.07
0.98
0.94
1.78
−0.42
−1.76
1.25
1.19
1.00
5.09
4.08
0.09




protein 5 (glucose-




regulated protein,




78 kDa)


GATM
NM_001482
glycine
15q15.1
Hs.75335
Mitochondria
1.08
0.80
0.61
2.62
−7.64
−18.24
1.23
0.78
0.79
7.34
−8.90
−9.41




amidinotransferase




(L-arginine:glycine




amidinotransferase)


CAT
NM_001752
catalase
11p13
Hs.395771
Oxidative
1.22
0.89
0.68
6.28
−3.94
−13.28
1.22
0.85
0.78
6.14
−4.94
−8.30







Stress


HADHB
NM_000183
hydroxyacyl-
2p23
Hs.269878
Mitochondria
1.11
0.88
0.71
3.71
−5.10
−14.11
1.22
0.87
0.74
6.30
−4.74
−10.47




Coenzyme A




dehydrogenase/3-




ketoacyl-Coenzyme




A thiolase/enoyl-




Coenzyme A




hydratase




(trifunctional




protein), beta




subunit


CCT3
NM_005998
chaperonin
1q23
Hs.1708
Chaperone
1.15
1.07
0.96
4.98
2.43
−1.67
1.22
1.04
1.03
5.97
1.34
0.96




containing TCP1,




subunit 3 (gamma)


DAD1
NM_001344
defender against
14q11–q12
Hs.82890
Apoptosis
1.13
1.00
0.88
4.36
0.15
−5.31
1.21
1.00
0.93
6.31
0.05
−2.86




cell death 1


HSPA2
NM_021979
heat shock 70 kDa
14q24.1
Hs.432648
Chaperone
1.36
0.68
0.69
9.00
−11.58
−12.22
1.21
0.69
1.01
5.95
−12.25
0.36




protein 2


NR4A1
NM_173157
nuclear receptor
12q13
Hs.1119
Mitochondria
0.83
0.86
1.17
−9.63
−8.30
9.19
0.83
0.90
1.15
−8.36
−5.21
7.06




subfamily 4, group




A, member 1


NAPG
NM_003826
N-ethylmaleimide-
18p11.21
Hs.370431
Mitochondria
0.94
1.07
1.69
−1.32
1.53
12.83
0.83
1.04
1.83
−3.06
0.60
10.97




sensitive factor




attachment protein,




gamma


MAPK1
NM_002745
mitogen-activated
22q11.21
Hs.324473
Mitochondria
0.85
0.99
1.12
−4.35
−0.35
3.34
0.82
0.96
1.07
−4.77
−1.10
1.82




protein kinase 1


DAD1
NM_001344
defender against
14q11–q12
Hs.82890
Apoptosis
1.04
1.04
1.39
0.94
0.91
8.60
0.79
1.07
1.46
−3.99
1.14
7.17




cell death 1


STIP1
NM_006819
stress-induced-
11q13
Hs.257827
Chaperone
1.17
1.16
0.97
4.50
4.51
−0.86
1.06
1.23
1.00
1.49
5.06
0.06




phosphoprotein 1




(Hsp70/Hsp90-




organizing protein)


DUSP1
NM_004417
dual specificity
5q34
Hs.171695
Oxidative
0.87
0.74
1.10
−4.00
−8.85
2.92
0.84
0.83
1.13
−4.06
−4.54
3.31




phosphatase 1


Stress


SLC25A13
NM_014251
solute carrier family
7q21.3
Hs.9599
Mitochondria
1.06
0.91
0.97
2.33
−3.94
−1.55
1.03
0.83
1.03
1.11
−6.61
1.10




25, member 13




(citrin)


PER2
NM_022817
period homolog 2
2q37.3
Hs.410692
Cycling
0.90
0.83
1.31
−3.22
−5.79
8.81
0.97
0.80
1.35
−0.76
−5.58
8.02




(Drosophila)


SST
NM_001048
somatostatin
3q28
Hs.12409
Apoptosis
1.16
0.88
3.34
3.92
−3.70
36.03
0.85
0.78
3.71
4.96
−7.89
45.04


DUSP6
NM_001946
dual specificity
12q22–q23
Hs.298654
Apoptosis
0.84
0.70
1.59
−4.15
−8.89
12.26
0.96
0.77
1.77
−0.93
−5.46
13.19




phosphatase 6


USP9Y
NM_004654
ubiquitin specific
Yq11.2
Hs.371255
26S
1.05
0.85
1.91
0.89
−3.32
13.69
1.17
0.75
2.20
2.39
−4.51
13.38




protease 9, Y-linked


proteasome




(fat facets-like,




Drosophila)


HSPA2
NM_021979
heat shock 70 kDa
14q24.1
Hs.432648
Chaperone
1.36
0.68
0.69
9.00
−11.58
−12.22
1.21
0.69
1.01
5.95
−12.25
0.36




protein 2


SEMA6A
NM_020796
sema domain,
5q23.1
Hs.443012
Apoptosis
0.84
0.74
0.68
−4.41
−8.25
−11.00
0.92
0.62
1.10
−1.70
−10.66
2.29




transmembrane




domain (TM), and




cytoplasmic




domain,




(semaphorin) 6A
























TABLE 21











QPCR

QPCR







Microarray
fold
Microarray
fold







fold change
change
fold change
change






Brain
(BPD v
(BPD
(MDD v
(MDD v


Accession
Symbol
Name
Cytoband
Region
Control)
v Control)
Control)
Control)























NM_001001935
ATP5A1
ATP synthase,
18q12–q21
DLPFC
1.203
1.587*
1.149
1.135




H+ transporting, mitochondrial F1




complex, alpha subunit, isoform 1,




cardiac muscle


NM_004047
ATP6V0B
ATPase, H+ transporting,
1p32.3
DLPFC
1.183
1.014
1.074
1.483*




lysosomal 21 kDa, V0 subunit c′′


NM_001696
ATP6V1E1
ATPase, H+ transporting,
22pter–q11.2
DLPFC
1.138
1.694*
1.081
0.939




lysosomal 31 kDa,




V1 subunit E isoform 1


NM_004458
ACSL4
Acyl-CoA synthetase long-chain
Xq22.3–q23
DLPFC
1.165
1.266*
1.105
1.095




family member 4


NM_174855
IDH3B
Isocitrate dehydrogenase 3
20p13
DLPFC
1.148
1.007
1.084
1.189*




(NAD+) beta


NM_133259
LRPPRC
Leucine-rich PPR-motif containing
2p21
DLPFC
1.218
1.62*
1.164
1.802*


NM_021107
MRPS12
Mitochondrial ribosomal protein S12
19q13.1–q13.2
DLPFC
1.091
1.140
1.110
1.276*


NM_007103
NDUFV1
NADH dehydrogenase (ubiquinone)
11q13
DLPFC
1.115
0.989
1.104
1.430*




flavoprotein 1, 51 kDa


NM_173157
NR4A1
Nuclear receptor subfamily 4,
12q13
DLPFC
0.832
0.584*
0.895
0.516*




group A, member 1


NM_000021
PSEN1
Presenilin 1 (Alzheimer disease 3)
14q24.3
DLPFC
1.081
1.306*
0.882
0.825


NM_001183
ATP6AP1
ATPase, H+ transporting,
Xq28
AnCg
1.134
1.448
1.112
1.385




lysosomal accessory protein 1


NM_021979
HSPA2
Heat shock 70 kDa protein 2
14q24.1
AnCg
1.360
2.552*
0.684
0.703


NM_174855
IDH3B
Isocitrate dehydrogenase 3
20p13
AnCg
1.136
0.998
1.099
0.985




(NAD+) beta


NM_133259
LRPPRC
Leucine-rich PPR-motif containing
2p21
AnCg
1.198
1.079
1.08
1.023


NM_007103
NDUFV1
NADH dehydrogenase (ubiquinone)
11q13
AnCg
1.169
1.480
1.148
1.107




flavoprotein 1, 51 kDa


NM_173157
NR4A1
Nuclear receptor subfamily 4,
12q13
AnCg
0.833
0.599*
0.859
0.484*




group A, member 1


NM_000021
PSEN1
Presenilin 1 (Alzheimer disease 3)
14q24.3
AnCg
1.109
1.460*
0.845
1.089


AK125435
PSMB1
Proteasome (prosome, macropain)
6q27
AnCg
1.271
1.615*
1.020
1.162




subunit, beta type, 1


NM_002812
PSMD8
Proteasome (prosome, macropain)
19q13.2
AnCg
1.155
1.358*
1.104
1.231*




26S subunit, non-ATPase, 8



























TABLE 22










QPCR
QPCR 








MtDNA



fold
fold



Encoded

Brain

change
change

start and

Com-
Hs.


Accession
Gene
Symbol
Region

BPD
MDD
Description
end bp
Table 1
plex
Unigene


























AY882398
MtATP6
ATP6
DLPFC
ATP synthase
0.853
0.876
Homo sapiens
8528 . . . 9208
Mito paper
V
Hs.4509






F0 subunit 6


isolate

Table 11









20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtATP6
ATP6
AnCg
ATP synthase
0.588*
0.721*
Homo sapiens
8528 . . . 9208
Mito paper
V
Hs.4509






F0 subunit 6


isolate

Table 11









20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtCO1
COX1
DLPFC
cytochrome
0.687
0.875
Homo sapiens
5905 . . . 7446
Mito paper
IV
Hs.4512






c oxidase,


isolate

Table 11






subunit I


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtCO1
COX1
AnCg
cytochrome
0.527*
0.808
Homo sapiens
5905 . . . 7446
Mito paper
IV
Hs.4512






c oxidase,


isolate

Table 11






subunit I


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtCO2
COX2
DLPFC
cytochrome
0.836
1.078
Homo sapiens
7587 . . . 8270
Mito paper
IV
Hs.4513






c oxidase,


isolate

Table 11






subunit II


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtCO2
COX2
AnCg
cytochrome
0.758#
0.895
Homo sapiens
7587 . . . 8270
Mito paper
IV
Hs.4513






c oxidase,


isolate

Table 11






subunit II


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtND1
ND1
DLPFC
NADH
0.563#
1.007
Homo sapiens
3308 . . . 4264
Mito paper
I
Hs.4535






dehydrogenase,


isolate

Table 11






subunit I


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtND1
ND1
AnCg
NADH
0.855
0.957
Homo sapiens
3308 . . . 4264
Mito paper
I
Hs.4535






dehydrogenase,


isolate

Table 11






subunit I


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtND2
ND2
DLPFC
NADH
0.539
0.921
Homo sapiens
4471 . . . 5514
Mito paper
I
Hs.4536






dehydrogenase,


isolate

Table 11






subunit II


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtND3
ND3
DLPFC
NADH
1.074
0.856
Homo sapiens
10060 . . . 10405
Mito paper
I
Hs.4537






dehydrogenase,


isolate

Table 11






subunit III


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtND3
ND3
AnCg
NADH
0.752#
0.918
Homo sapiens
10060 . . . 10405
Mito paper
I
Hs.4537






dehydrogenase,


isolate

Table 11






subunit III


20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.


AY882398
MtATP6
ATP6
DLPFC
ATP synthase
0.853
0.876
Homo sapiens
8528 . . . 9208
Mito paper
V
Hs.4509






F0 subunit 6


isolate

Table 11









20_U5a1(Tor13)

(mt)









mitochondrion,









complete genome.





*significant at p < 0.05 two-tailed


#trend p < 0.1 two-tailed















TABLE 23





Primer
Forward
Reverse


















VASE
5′-GACCCCATTCCCTCCATCAC-3′
5′-GGCTACGCACCACCATGTG-3′



 


Exon a
5′′-GACGCAGCCAGTCCATAGC-3′
*1


 


Exon b
5′-CGTCTACCCCTGTTCCATTGTC-3′
5′-TCTGGTGGAGACAATGGAACAG-3′


 


Exon c
5′-TCCTGCCCTTGCAACCA-3′
5′-GGTTGCAAGGGCAGGAAGA-3′


 


SEC exon
5′-CCAAGCTGGTCTTCATAATGCTCTA-3′
5′TTTGATGCTTGAACACTATGAACATG-3′


 


Exon 3
5′-GGCGGCGCTCAATGG-3′
*2


 


Exon 8
*3
5′-GATCAGGTTCACTTTAATAGAGTTTCCA-3′


 


SNP9 for sequencing
5′-CGCAGCCAGTCCGTAAGTAAAG-3′
5′-AAGCTGGACCGGCTACTAGGA-3′
















TABLE 24







Tests For Genotypic Association (Risk Allele 1)











Heterozygous
Homozygous
Allele Positivity














Odds Ratio [C.I.]
χ2 (p-value)
Odds Ratio [C.I.]
χ2 (p-value)
Odds Ratio [C.I.]
χ2 (p-value)













SNP 9
A/A<->C/A
C/C<->A/A
[C/C + C/A]<->A/A













SZ
0.054 [0.003-1.16]
 6.6 (0.01)
9.57 [0.47-193.92]
3.86 (0.049)
0.084 [0.004-1.67]
4.88 (0.027)










SNP b
C/C<->T/C
T/T<->C/C
[T/T + T/C]<->C/C













BPD
 4.05 [1.16-14.12]
5.33 (0.02)
2.97 [0.78-11.30]
2.65 (0.103)
 3.66 [1.08-11.30]
4.81 (0.028)




















TABLE 25








Haplotype

Frequency (Odds Ratio)













SNP 9 − SNP b
Control
BPD*
SZ #






C − T
0.2
0.19 (0.95)
0.31 (1.54)



C − (T/C)
0.31
0.46 (1.48)
0.37 (1.19)



(C/A) − (T/C)
0.22
0.21 (0.95)
0.11 (0.50)













p (value)



*BPD vs Control
<0.0001



# SZ vs Control
<0.0001



SZ vs BPD
0.0003




















TABLE 26









Genotype
Allele Counts
p (Fisher's











Polymorphism
n
Counts (Frequency)
(Frequency)
exact test)

















SNP 9

C/C
C/A
A/A
C
A






Control
55
33 (0.60)
22 (0.40)
 0 (0)
 88 (0.80)
22 (0.20)


BPD
70
47 (0.67)
23 (0.33)
 0 (0)
117 (0.84)
23 (.16)
0.466


SZ
35
24 (0.69)
 8 (0.23)
 3 (0.09)
 56 (0.80)
14 (0.20)
1


BPD + SZ
105
71 (0.68)
31 (0.30)
 3 (0.02)
173 (0.82)
37 (0.18)
0.602





SNP b

T/T
T/C
C/C
T
C





Control
55
16 (0.29)
29 (0.53)
10 (0.18)
 61 (0.55)
49 (0.45)


BPD
70
19 (0.27)
47 (0.67)
 4 (0.06)
 85 (0.61)
55 (.39)
0.402


SZ
35
13 (0.37)
19 (0.54)
 3 (0.09)
 45 (0.64)
25 (0.36)
0.24


BPD + SZ
105
32 (0.30)
66 (0.63)
 7 (0.07)
130 (0.62)
80 (0.38)
0.264




















TABLE 27





SNP
Genotype
Splice Variant
BPD vs C
MDD vs C







SNP 9
C/C
a-b-c
0.052 ↑



SNP 6
G/C
b-c-SEC

0.05 ↑ 


SNP b
T/T
VASE (−)

0.056 ↑


SNP b
T/C
c-SEC
0.013 ↓



SNP b
T/C
NCAM1 Ct Q-PCR

0.053 ↑




















TABLE 28.1





UG Cluster
Symbol
Gene Name
Cytoband
fc_AnCg



















Hs.549038



1.919839205


Hs.547062

Transcribed locus

1.773637669


Hs.483454
CNN3
Calponin 3, acidic
1p22-p21
1.71410127


Hs.534365
ZNF43
Zinc finger protein 43 (HTF6)
19p13.1-p12
1.653473114


Hs.534314
EIF5A
Eukaryotic translation initiation factor 5A
17p13-p12
1.616760481


Hs.427236

Transcribed locus

1.57275662


Hs.336957
ZNF479
Zinc finger protein 479
7p11.2
1.561114027


Hs.534385
THOC4
THO complex 4
17q25.3
1.545094715


Hs.114084
ENPP7
Ectonucleotide pyrophosphatase/phosphodiesterase 7
17q25.3
1.490770143


Hs.315369
AQP4
Aquaporin 4
18q11.2-q12.1
1.478027231


Hs.370410
KIAA1145
KIAA1145 protein
12q22
1.435347843


Hs.75914
RNP24
Coated vesicle membrane protein
12q24.31
1.419831899


Hs.406708
ILT7
Leukocyte immunoglobulin-like receptor, subfamily A
19q13.4
1.410439598




(without TM domain), member 4




Hs.234249
MAPK8IP1
Mitogen-activated protein kinase 8 interacting protein 1
11p12-p11.2
1.371562585


Hs.534525
LOC114984
Hypothetical protein BC014089
16p13.3
1.368785293


Hs.212838
A2M
Alpha-2-macroglobulin
12p13.3-p12.3
1.365561844


Hs.402752
TAF15
TAF15 RNA polymerase II, TATA box binding protein
17q11.1-q11.2
1.362205401




(TBP)-associated factor, 68 kDa




Hs.535415

Ig rearranged gamma-chain mRNA, subgroup VH2, V-D-J region
1.355666889



Hs.467138

Transcribed locus, moderately similar to XP_507997.1
1.351924341





similar to Kv channel interacting protein 2 isoform 4; A-type






potassium channel modulatory protein 2; cardiac voltage






gated potassium channel modulatory subunit; Kv channel-






interacting protein 2 [Pan tr




Hs.513600

Transcribed locus

1.347512479


Hs.380218

Transcribed locus

1.327051286


Hs.458607
NOPE
Likely ortholog of mouse neighbor of Punc E11
15q22.31
1.324220424


Hs.42034
TCP10L
T-complex 10 (mouse)-like
21q22.11
1.322514626


Hs.521286
RARRES2
Retinoic acid receptor responder (tazarotene induced) 2
7q36.1
1.31025151


Hs.25422

CDNA FLJ42519 fis, clone BRACE3000787

1.309835897


Hs.293379

Transcribed locus

1.307793259


Hs.356766

Similar to RPE-spondin
20q13.13
1.290259345


Hs.379010
PSCA
Prostate stem cell antigen
8q24.2
1.287232595


Hs.511757
GJB6
Gap junction protein, beta 6 (connexin 30)
13q11-q12.1
1.287226239


Hs.59106
CGRRF1
Cell growth regulator with ring finger domain 1
14q22.2
1.284552431


Hs.83916
NDUFA5
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 13 kDa
7q32
1.283862892


Hs.119878
FLJ34389
Hypothetical protein FLJ34389
16q22.3
1.28271805


Hs.494261
PSAT1
Phosphoserine aminotransferase 1
9q21.2
1.276727208


Hs.75969
PNRC1
Proline-rich nuclear receptor coactivator 1
6q15
1.273555895


Hs.467273
CACNG8
Calcium channel, voltage-dependent, gamma subunit 8
19q13.4
1.270119391


Hs.280805
MGC20579
Hypothetical protein MGC20579
13q34
1.269164507


Hs.145480

Transcribed locus

1.266189386


Hs.511454
PLXNA4
Plexin A4
7q32.3
1.263673467


Hs.80132
SNX15
Sorting nexin 15
11q12
1.26259913


Hs.514819
AP2B1
Adaptor-related protein complex 2, beta 1 subunit
17q11.2-q12
1.250901951


Hs.385772
LOC283914
Hypothetical protein LOC283914
16p11.1
1.247941693


Hs.151536
RAB13
RAB13, member RAS oncogene family
1q21.2
1.247404054


Hs.548424

Transcribed locus

1.245303451


Hs.519930
C6orf62
Chromosome 6 open reading frame 62
6p22.2
1.233111115


Hs.45140
TMEM35
Transmembrane protein 35
Xq22.1
1.232645678


Hs.505295
MADP-1
MADP-1 protein
12q12
1.226870897


Hs.513883
PELP1
Proline-, glutamic acid-, leucine-rich protein 1
17p13.2
1.224880728


Hs.474836
LOC387593
TPTE/TPIP pseudogene
22q13
1.223415544


Hs.115284
ZNF213
Zinc finger protein 213
16p13.3
1.223309419


Hs.369624
15E1.2
Hypothetical protein 15E1.2
12q24.31
1.223144605


Hs.467960
RAB10
RAB10, member RAS oncogene family
2p23.3
1.222273799


Hs.533282
NONO
Non-POU domain containing, octamer-binding
Xq13.1
1.219220007


Hs.181272
PKD2
Polycystic kidney disease 2 (autosomal dominant)
4q21-q23
1.212879872


Hs.80720
GAB1
GRB2-associated binding protein 1
4q31.21
1.211569918


Hs.123464
P2RY5
Purinergic receptor P2Y, G-protein coupled, 5
13q14
1.210914564


Hs.507185
ZFPM1
Zinc finger protein, multitype 1
16q24.2
1.210160133


Hs.5324
C2orf25
Chromosome 2 open reading frame 25
2q23.3
1.209212561


Hs.374847
LOC400794
Hypothetical gene supported by BC030596
1q23.2
1.208951568


Hs.517792
C3orf10
Chromosome 3 open reading frame 10
3p25.3
1.208760837


Hs.517352
PRODH
Proline dehydrogenase (oxidase) 1
22q11.21
1.206253745


Hs.483561
ORF1-FL49
Putative nuclear protein ORF1-FL49
5q31.2
1.205748855


Hs.75640
NPPA
Natriuretic peptide precursor A
1p36.21
1.20542617


Hs.491695
UBE2V2
Ubiquitin-conjugating enzyme E2 variant 2
8q11.21
1.204850064


Hs.335057
NEDD5
Neural precursor cell expressed, developmentally
2q37
1.202774491




down-regulated 5




Hs.28280
SLC35F4
Solute carrier family 35, member F4
14q22.2
1.201026204




















TABLE 28.2





UG Cluster
Symbol
Gene Name
Cytoband
fc_AnCg



















Hs.134974
GAP43
Growth associated protein 43
3q13.1-q13.2
0.833001805


Hs.444637
LRP8
Low density lipoprotein receptor-related protein 8, apolipoprotein
1p34
0.832591468




e receptor




Hs.268849
GLO1
Glyoxalase I
6p21.3-p21.1
0.832479968


Hs.435952
CDK5RAP1
CDK5 regulatory subunit associated protein 1
20pter-q11.23
0.831820461


Hs.360940
dJ222E13.1
Kraken-like
22q13
0.828943053


Hs.500721
MMS19L
MMS19-like (MET18 homolog, S. cerevisiae)
10q24-q25
0.827060597


Hs.200285
TCF4
Transcription factor 4
18q21.1
0.826908526


Hs.2890
PRKCG
Protein kinase C, gamma
19q13.4
0.82630472


Hs.153661

Transcribed locus

0.825857073


Hs.483924
MRPL22
Mitochondrial ribosomal protein L22
5q33.1-q33.3
0.825300862


Hs.210385
HERC1
Hect (homologous to the E6-AP (UBE3A) carboxyl terminus)
15q22
0.82523312




domain and RCC1 (CHC1)-like domain (RLD) 1




Hs.353454
FLJ10276
Hypothetical protein FLJ10276
1p35.1
0.825093453


Hs.157234

MRNA; cDNA DKFZp547A0515 (from clone DKFZp547A0515)

0.824586185


Hs.75667
SYP
Synaptophysin
Xp11.23-p11.22
0.824282156


Hs.451353

Homo sapiens, clone IMAGE: 5288537, mRNA

0.823552515


Hs.514373
MTMR4
Myotubularin related protein 4
17q22-q23
0.823152304


Hs.102696
MCTS1
Malignant T cell amplified sequence 1
Xq22-q24
0.822759185


Hs.445503
SYN2
Synapsin II
3p25
0.822588683


Hs.524094
PS1D
Putative S1 RNA binding domain protein
1p35.2
0.822570466


Hs.522668
UBQLN2
Ubiquilin 2
Xp11.23-p11.1
0.821156426


Hs.517148
TH1L
TH1-like (Drosophila)
20q13
0.821107397


Hs.380334
ZNF148
Zinc finger protein 148 (pHZ-52)
3q21
0.821027253


Hs.534575
MGC2198
Hypothetical protein MGC2198
5q35.2
0.819426558


Hs.47546
C6orf70
Chromosome 6 open reading frame 70
6q27
0.819049461


Hs.25601
CHD3
Chromodomain helicase DNA binding protein 3
17p13.1
0.818740301


Hs.460978
APPBP1
Amyloid beta precursor protein binding protein 1, 59 kDa
16q22
0.818504577


Hs.448851
USP6
Ubiquitin specific protease 6 (Tre-2 oncogene)
17q11
0.818420142


Hs.532755
GTL3
Likely ortholog of mouse gene trap locus 3
16q21
0.816593983


Hs.98510
WDR44
WD repeat domain 44
Xq24
0.815070227


Hs.189119
CXXC5
CXXC finger 5
5q31.2
0.814065874


Hs.134060
FNBP1L
Formin binding protein 1-like
1p22.1
0.813237448


Hs.549821

Data not found

0.812556603


Hs.78944
RGS2
Regulator of G-protein signalling 2, 24 kDa
1q31
0.811942329


Hs.535060

LOC441385
9p24.1
0.811837917


Hs.549166

Data not found

0.811572937


Hs.224418

Transcribed locus

0.811556308


Hs.5258
MAGED1
Melanoma antigen, family D, 1
Xp11.23
0.811035568


Hs.537449

Transcribed locus

0.810141577


Hs.515545
TBC1D17
TBC1 domain family, member 17
19q13.33
0.809453648


Hs.268122
LOC51321
Hypothetical protein LOC51321
17q24.2
0.808975121


Hs.502910
NKIRAS2
NFKB inhibitor interacting Ras-like 2
17q21.2
0.808919755


Hs.471876
ING5
Inhibitor of growth family, member 5
2q37.3
0.806159689


Hs.198612
GPR51
G protein-coupled receptor 51
9q22.1-q22.3
0.806097527


Hs.401509
RBM10
RNA binding motif protein 10
Xp11.23
0.806076882


Hs.380857
TD-60
RCC1-like
1p36.13
0.805526827


Hs.49582
PPP1R12A
Protein phosphatase 1, regulatory (inhibitor) subunit 12A
12q15-q21
0.805427552


Hs.515162
CALR
Calreticulin
19p13.3-p13.2
0.804252658


Hs.444558
KHDRBS3
KH domain containing, RNA binding, signal transduction
8q24.2
0.803823019




associated 3




Hs.317632
CDH18
Cadherin 18, type 2
5p15.2-p15.1
0.803613164


Hs.471104
NOP5/NOP58
Nucleolar protein NOP5/NOP58
2q33.1
0.802352232


Hs.348526
LOC474358
Hypothetical BC042079 locus
10q23-q25
0.802291667


Hs.475018
TCF20
Transcription factor 20 (AR1)
22q13.3
0.801897074


Hs.463074
ATP6V0A1
ATPase, H+ transporting, lysosomal V0 subunit a isoform 1
17q21
0.801766875


Hs.28144
FSD1
Fibronectin type 3 and SPRY domain containing 1
19p13.3
0.800329999


Hs.32309
INPP1
Inositol polyphosphate-1-phosphatase
2q32
0.796446965


Hs.512973
HSPC121
Butyrate-induced transcript 1
15q22.2
0.796376497


Hs.13245
LPPR4
Plasticity related gene 1
1p21.3
0.794472742


Hs.199743
ME3
Malic enzyme 3, NADP(+)-dependent, mitochondrial
11cen-q22.3
0.794075417


Hs.523755
FLRT1
Fibronectin leucine rich transmembrane protein 1
11q12-q13
0.792664481


Hs.515785
BLVRB
Biliverdin reductase B (flavin reductase (NADPH))
19q13.1-q13.2
0.792511112


Hs.437277
MGAT4B
Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-
5q35
0.790721678




acetylglucosaminyltransferase, isoenzyme B




Hs.387982

CDNA clone IMAGE: 5261489, partial cds

0.789879979


Hs.136164
TSPYL2
TSPY-like 2
Xp11.2
0.788609957


Hs.332847
CRIM1
Cysteine-rich motor neuron 1
2p21
0.786568887


Hs.348493
GPRASP2
G protein-coupled receptor associated sorting protein 2
Xq22.1
0.786263129


Hs.7736
MRPL27
Mitochondrial ribosomal protein L27
17q21.3-q22
0.785613917


Hs.177275
ANKRD6
Ankyrin repeat domain 6
6q14.2-q16.1
0.783389812


Hs.124015
HAGHL
Hydroxyacylglutathione hydrolase-like
16p13.3
0.782617204


Hs.79322
QARS
Glutaminyl-tRNA synthetase
3p21.3-p21.1
0.781931967


Hs.158748
SLC35F3
Solute carrier family 35, member F3
1q42.2
0.780681609


Hs.158460
CDK5R2
Cyclin-dependent kinase 5, regulatory subunit 2 (p39)
2q35
0.779713574


Hs.337730
LCMT1
Leucine carboxyl methyltransferase 1
16p12.3-16p12.1
0.777655555


Hs.282998
RBM9
RNA binding motif protein 9
22q13.1
0.775799713


Hs.60300
ZNF622
Zinc finger protein 622
5p15.1
0.775473024


Hs.405590
EIF3S6
Eukaryotic translation initiation factor 3, subunit 6 48 kDa
8q22-q23
0.775166314


Hs.373952
CAMTA2
Calmodulin binding transcription activator 2
17p13.2
0.775135303


Hs.528187

Hypothetical gene supported by AK096649
2q33.1
0.774877993


Hs.536326

Transcribed locus

0.774173869


Hs.532231
COPG2
Coatomer protein complex, subunit gamma 2
7q32
0.773663441


Hs.114169
LRRTM2
Leucine rich repeat transmembrane neuronal 2
5q31.3
0.773056562


Hs.496267
IGBP1
Immunoglobulin (CD79A) binding protein 1
Xq13.1-q13.3
0.771399953


Hs.190722
HSPC142
HSPC142 protein
19p13.11
0.770969106


Hs.130197
KIAA1889
KIAA1889 protein
8q12.1
0.770498302


Hs.436446
ARMET
Arginine-rich, mutated in early stage tumors
3p21.1
0.769942554


Hs.381300
MGC57858
Hypothetical protein MGC57858
6p21.31
0.769698647


Hs.33191
UNC5A
Unc-5 homolog A (C. elegans)
5q35.2
0.769489374


Hs.350065
PLXNA2
Plexin A2
1q32.2
0.767903428


Hs.48372

Full length insert cDNA clone YZ87G11

0.766810973


Hs.3797
RAB26
RAB26, member RAS oncogene family
16p13.3
0.765394204


Hs.21925

Transcribed locus

0.764561674


Hs.336588
LOC147670
Hypothetical protein LOC147670
19q13.43
0.763351376


Hs.78466
PSMD8
Proteasome (prosome, macropain) 26S subunit, non-ATPase, 8
19q13.2
0.76234581


Hs.121520
AMIGO2
Amphoterin induced gene 2
12q13.11
0.760653807


Hs.443731
USP8
Ubiquitin specific protease 8
15q21.2
0.75760815


Hs.171501
USP11
Ubiquitin specific protease 11
Xp11.23
0.755609225


Hs.187861
THRB
Thyroid hormone receptor, beta (erythroblastic leukemia viral (verb-
3p24.3
0.755503889




a) oncogene homolog 2, avian)




Hs.272284
SLITRK4
SLIT and NTRK-like family, member 4
Xq27.3
0.755411535


Hs.509736
HSPCB
Heat shock 90 kDa protein 1, beta
6p12
0.755057388


Hs.188594

Transcribed locus

0.755026908


Hs.497806
MARK1
MAP/microtubule affinity-regulating kinase 1
1q41
0.751792399


Hs.549761

Data not found

0.749944


Hs.479867
CENPC1
Centromere protein C 1
4q12-q13.3
0.749713558


Hs.135736
NGL-1
Netrin-G1 ligand
11p12
0.747993997


Hs.463466
CA10
Carbonic anhydrase X
17q21
0.746516358


Hs.143587

Transcribed locus

0.745018326


Hs.549196

Data not found

0.744120055


Hs.534913

Hypothetical gene supported by BC019717
16p11.2
0.743499122


Hs.523550
ZNF364
Zinc finger protein 364
1q21.1
0.742420643


Hs.90242

Homo sapiens, clone IMAGE: 4796172, mRNA

0.741848633


Hs.475150
KIAA0767
KIAA0767 protein
22q13.31
0.741559246


Hs.530698
CHD8
Chromodomain helicase DNA binding protein 8
14q11.2
0.738476834


Hs.55879
ABCC10
ATP-binding cassette, sub-family C (CFTR/MRP), member 10
6p21.1
0.738455293


Hs.323537
FLJ12953
Hypothetical protein FLJ12953 similar to Mus musculus D3Mm3e
2p13.1
0.737391028


Hs.301296

CDNA: FLJ23131 fis, clone LNG08502

0.737354551


Hs.502461
DGKZ
Diacylglycerol kinase, zeta 104 kDa
11p11.2
0.734852399


Hs.471096
ALS2
Amyotrophic lateral sclerosis 2 (juvenile)
2q33.1
0.73175125


Hs.92732
PDZK4
PDZ domain containing 4
Xq28
0.727016153


Hs.537841

Transcribed locus

0.726748437


Hs.7744
NDUFV1
NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa
11q13
0.726496223


Hs.100890
RPRM
Reprimo, TP53 dependant G2 arrest mediator candidate
2q23.3
0.719810824


Hs.6132
CPNE6
Copine VI (neuronal)
14q11.2
0.718586854


Hs.412019
C6orf80
Chromosome 6 open reading frame 80
6q23.1-q24.1
0.715749314


Hs.119594
CIT
Citron (rho-interacting, serine/threonine kinase 21)
12q24
0.712998565


Hs.518460
AP2M1
Adaptor-related protein complex 2, mu 1 subunit
3q28
0.709632072


Hs.65425
CALB1
Calbindin 1, 28 kDa
8q21.3-q22.1
0.70924145


Hs.479116
SH3TC1
SH3 domain and tetratricopeptide repeats 1
4p16.1
0.704347818


Hs.173859
FZD7
Frizzled homolog 7 (Drosophila)
2q33
0.697933574


Hs.363137
ACAT2
Acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme
6q25.3-q26
0.695447549




A thiolase)




Hs.153648
PPFIA4
Protein tyrosine phosphatase, receptor type, f polypeptide
1q32.1
0.694615615




(PTPRF), interacting protein (liprin), alpha 4




Hs.524071

Transcribed locus, strongly similar to XP_084672.3 similar to

0.691986755




CDNA sequence BC021608 [Homo sapiens]




Hs.506784
LNK
Lymphocyte adaptor protein
12q24
0.688023619


Hs.516617
SATB2
SATB family member 2
2q33
0.681642635


Hs.23406
KCTD4
Potassium channel tetramerisation domain containing 4
13q14.12
0.676435668


Hs.220950
FOXO3A
Forkhead box O3A
6q21
0.669990152


Hs.370549
BCL11A
B-cell CLL/lymphoma 11A (zinc finger protein)
2p16.1
0.654002694


Hs.483239
ALDH7A1
Aldehyde dehydrogenase 7 family, member A1
5q31
0.651849727


Hs.445733
GSK3B
Glycogen synthase kinase 3 beta
3q13.3
0.647879909


Hs.268515
MN1
Meningioma (disrupted in balanced translocation) 1
22q11
0.64462122


Hs.319503
PTCHD1
Patched domain containing 1
Xp22.11
0.644497163


Hs.106511
PCDH17
Hypothetical protein LOC144997
13q21.1
0.644270897


Hs.91448
DUSP14
Dual specificity phosphatase 14
17q12
0.639891125


Hs.448041
FLJ32363
FLJ32363 protein
5p12
0.633100428


Hs.22584
PDYN
Prodynorphin
20pter-p12
0.630525299


Hs.215839
DLG2
Discs, large homolog 2, chapsyn-110 (Drosophila)
11q21
0.620144715


Hs.11899
HMGCR
3-hydroxy-3-methylglutaryl-Coenzyme A reductase
5q13.3-q14
0.619486994


Hs.23539

CDNA FLJ42249 fis, clone TKIDN2007667

0.614085673


Hs.314436
NEDL2
NEDD4-related E3 ubiquitin ligase NEDL2
2q32.3
0.605505392


Hs.490294
KIAA1549
KIAA1549 protein
7q34
0.548338996


Hs.518469
FLJ10560
Hypothetical protein FLJ10560
3q27.3
0.498445319


Hs.546322
NOL4
Nucleolar protein 4
18q12
0.491050809


Hs.282177
PIP5K1C
Phosphatidylinositol-4-phosphate 5-kinase, type I, gamma
19p13.3
0.490622069


Hs.2785
KRT17
Keratin 17
17q12-q21
0.474185311


Hs.536506

Transcribed locus

0.467413109


Hs.435001
KLF10
Kruppel-like factor 10
8q22.2
0.463636382


Hs.537539

Transcribed locus

0.358666831
















TABLE 29







v-ATPase Human MDD vs Monkey stressed Array Results















Human T-test
Human T-test
Monkey





Affy HIP
illu HIP
Midlife Stress


UGRepAcc
Name
Symbol
t2hcmdd.affy
tHCMdd.illu
change















BQ230447
ATPase, H+ transporting, lysosomal
ATP6V0E
−2.8565
−2.2254
−1.07



9 kDa, V0 subunit e


AF245517
ATPase, H+ transporting, lysosomal
ATP6V0A4
−1.6935
−1.5208
1.01



V0 subunit a isoform 4


NM_012463
ATPase, H+ transporting, lysosomal
ATP6V0A2
−0.8322
−1.6802
−1.05



V0 subunit a isoform 2


CR607789
ATPase, H+ transporting, lysosomal
ATP6V1G1
−0.0535
−0.0440
−1.08



13 kDa, V1 subunit G isoform 1


AK127853
ATPase, H+ transporting, lysosomal
ATP6V1B1
0.3202
−0.8313
1.02



56/58 kDa, V1 subunit B, isoform 1



(Renal tubular acidosis with deafness)


BF214530
ATPase, H+ transporting, lysosomal
ATP6V1E1
1.3703
1.9643
−1.01



31 kDa, V1 subunit E isoform 1


AK024101
ATPase, H+ transporting, lysosomal
ATP6V1D
1.6380
2.6589
1.09



34 kDa, V1 subunit D


NM_001690
ATPase, H+ transporting, lysosomal
ATP6V1A
1.7837
2.4222
1.15



70 kDa, V1 subunit A


NM_001695
ATPase, H+ transporting, lysosomal
ATP6V1C1
1.7882
1.0692
−1.01



42 kDa, V1 subunit C, isoform 1


AK128641
ATPase, H+ transporting, lysosomal
ATP6V0D1
1.8249
2.4330
1.03



38 kDa, V0 subunit d isoform 1


BC053601
ATPase, H+ transporting, lysosomal
ATP6V0B
1.8268
−0.2217
1.10



21 kDa, V0 subunit c′′


AK127505
ATPase, H+ transporting, lysosomal
ATP6V0C
2.5856
2.2705
−1.04



16 kDa, V0 subunit c


NM_001693
ATPase, H+ transporting, lysosomal
ATP6V1B2
2.6827
2.6120
1.04



56/58 kDa, V1 subunit B, isoform 2


AK125927
ATPase, H+ transporting, lysosomal
ATP6V0A1
3.3021
1.2269
1.04



V0 subunit a isoform 1

























TABLE 30





Probe Set



AII AD
AII AD


AII CUS +
AII CUS +


Name
Identifier
LocusLink
Name
H2O Ave
CUS Ave
CUS T
CUS FC
AD T
AD FC
























1370043_at
NM_031753
79559
activated leukocyte
8.554991091
8.737278
0.305517
1.134681103
−0.337997115
0.87551622





cell adhesion





molecule


1375424_at
BE107525
64040
aldehyde
7.7269596
7.985884
0.253566
1.196585931
−0.275888751
0.815851533





dehydrogenase





family 9, subfamily





A1


1370176_at
BG378620
171086
amyotrophic lateral
8.289171999
8.644384
0.549287
1.279173789
−0.422598222
0.816014485





sclerosis 2





(juvenile)





chromosome





region, candidate 3


1370050_at
NM_053311
29598
ATPase, Ca++
10.02156406
10.47301
0.407594
1.367412006
−0.303601287
0.776657575





transporting,





plasma membrane 1


1367585_a_at
M28647
24211
ATPase, Na+K+
10.86677482
10.70967
−0.27986
0.896825718
0.365553735
1.117986929





transporting, alpha 1


1398781_at
NM_053884
116664
ATPase, vacuolar,
9.794708887
9.635878
−0.40588
0.895750971
0.501130634
1.131514226





14 kD


1367595_s_at
NM_012512
24223
Beta-2-
10.16168978
9.88582
−0.59779
0.825952261
0.384629381
1.135862492





microglobulin


1370074_at
NM_057196
117542
brain-specific
8.394268852
8.230674
−0.55098
0.892797502
0.467062082
1.118839281





angiogenesis





inhibitor 1-





associated protein 2


1369993_at
NM_133605
171140
calcium/calmodulin-
8.242780231
8.480603
0.338888
1.179211766
−0.292444973
0.860882267





dependent





protein kinase





(CaM kinase) II





gamma


1389824_at
BF404381
25400
calcium/calmodulin-
7.810218827
7.950381
0.416069
1.102029271
−0.470849794
0.896960966





dependent





protein kinase II





alpha subunit


1398251_a_at
NM_021739
24245
calcium/calmodulin-
8.228441025
8.843395
0.474736
1.531509499
−0.367083116
0.701179029





dependent





protein kinase II





beta subunit


1367462_at
U10861
29156
calpain, small
9.116789268
8.937121
−0.50886
0.882905842
0.648775676
1.158940404





subunit 1


1389876_at
BE111167
287005
CaM-kinase II
8.411415385
8.683458
0.388539
1.207516219
−0.331393094
0.833810939





inhibitor alpha


1370853_at
AA858621
287005
CaM-kinase II
9.552520138
9.824481
0.484035
1.207447775
−0.302368972
0.879487359





inhibitor alpha


1369215_a_at
NM_012836
25306
carboxypeptidase D
7.60221365
7.851067
0.409687
1.188262417
−0.318247147
0.865883498


1389974_at
BF555171
116549
casein kinase II,
7.070655253
7.33655
0.576709
1.20238131
−0.509595002
0.849661157





alpha 1





polypeptide


1387436_at
NM_022616
64551
CDC10 (cell
8.264076026
7.838606
−0.82282
0.744596339
0.558756443
1.170398833





division cycle 10,





S. cerevisiae,





homolog)


1370922_at
L15011
29145
cortexin
10.52034023
10.29252
−0.39042
0.853925826
0.642216784
1.297259097


1368059_at
NM_053955
117024
crystallin, mu
9.15043777
8.934658
−0.55932
0.861080678
0.40726188
1.109586413


1370438_at
AF037071
192363
C-terminal PDZ
8.478106643
8.941752
0.528156
1.37902155
−0.286782718
0.819872562





domain ligand of





neuronal nitric





oxide synthase


1370810_at
L09752
64033
cyclin D2
7.552338207
7.824805
0.627432
1.207871164
−0.510232497
0.856351578


1370180_at
AA891213
94267
diphosphoinositol
9.720407691
9.551001
−0.32062
0.889208564
0.33907154
1.112564538





polyphosphate





phosphohydolase





type II


1399090_at
AA944459
252902
dynein,
8.708970325
9.160194
0.592983
1.36719938
−0.611477208
0.713171499





cytoplasmic, light





intermediate chain 1


1370048_at
NM_053936
116744
endothelial
8.190874036
8.434857
0.460008
1.184257781
−0.276403406
0.88926266





differentiation,





lysophosphatidic





acid G-protein-





coupled receptor, 2


1370341_at
AF019973
24334
enolase 2, gamma
10.25966095
10.09239
−0.27444
0.890528386
0.511287186
1.226632739


1367958_at
NM_024397
79249
eps8 binding
7.501336434
7.734993
0.390605
1.175811622
−0.214182288
0.899358171





protein (e3B1),





alternatively





spliced


1373067_at
AI102738

ESTs
9.097780684
9.795279
0.560829
1.621690153
−0.394405591
0.691167336


1375687_at
BE097926

ESTs
9.102908418
9.589148
0.67554
1.400788553
−0.668809291
0.710828697


1375343_at
BE116572

ESTs
9.636710979
10.05569
0.637477
1.336979858
−0.600017235
0.721789025


1390722_at
AW531272

ESTs
8.052433351
8.445177
0.563541
1.312887882
−0.564580433
0.737665796


1371776_at
AA819268

ESTs
8.792612117
9.070286
0.439984
1.21223885
−0.60941328
0.751754214


1377029_at
AI235414

ESTs
7.519789299
8.066421
0.538435
1.460671268
−0.347221628
0.758851202


1374002_at
AI045904

ESTs
7.891491864
8.3451
0.66405
1.369460535
−0.489475041
0.773784892


1372183_at
AI230596

ESTs
7.499883481
7.870917
0.59093
1.293279024
−0.538050749
0.787917813


1390100_s_at
BG371810

ESTs
8.847513531
9.335307
0.623353
1.402298615
−0.383584226
0.796768397


1376463_at
AA955579

ESTs
8.580618929
8.884818
0.477979
1.234732891
−0.512299476
0.797964077


1380433_at
AI229240

ESTs
8.198656524
8.544649
0.807649
1.271025354
−0.703481364
0.812645109


1376911_at
BM386385

ESTs
8.998969791
9.304256
0.378751
1.235663347
−0.292496741
0.834798828


1374276_at
BE104102

ESTs
8.042219162
8.352924
0.479598
1.24031378
−0.357883889
0.843306633


1393268_at
AI071071

ESTs
7.760844879
8.008586
0.670383
1.187346647
−0.604731486
0.847734973


1385889_at
AA893212

ESTs
7.456567924
7.681123
0.541819
1.168417012
−0.524932857
0.856389756


1388985_at
AI012869

ESTs
10.3902729
10.71161
0.771777
1.249486875
−0.374963851
0.861337025


1375144_at
BM388843

ESTs
9.305640348
9.77995
0.689094
1.389253394
−0.247358915
0.862096412


1375850_at
BG371810

ESTs
10.32943696
10.5907
0.646492
1.198530654
−0.463027833
0.863301098


1376685_at
AW532489

ESTs
7.03405558
7.31854
0.585335
1.217975253
−0.362402037
0.873963777


1375538_at
AI230737

ESTs
7.60108064
7.77676
0.590257
1.129495994
−0.611495294
0.87898368


1377232_at
BF406608

ESTs
7.50385238
7.649913
0.456061
1.106543988
−0.555511902
0.885897068


1374485_at
AI137762

ESTs
7.635036986
7.803592
0.478724
1.123932187
−0.480827091
0.88597741


1389104_s_at
BF388420

ESTs
7.793627056
7.97877
0.44046
1.136929557
−0.340035773
0.897093342


1372790_at
BG671530

ESTs
9.913991148
9.6554
−0.42594
0.835903569
0.333317197
1.121671745


1388738_at
AI411227

ESTs
9.013127784
8.836253
−0.32841
0.884617394
0.421781411
1.134002323


1389600_at
AW524433

ESTs
9.341707252
9.136027
−0.31744
0.867129737
0.294052169
1.146455757


1388195_at
AW140475

ESTs
8.841214015
8.623431
−0.48629
0.859885815
0.493109601
1.177343855


1389867_at
BI281086

ESTs
9.622618423
9.295352
−0.44041
0.797045168
0.527626149
1.325364171


1371977_at
BG381477

ESTs, Highly
8.22534466
7.988656
−0.43238
0.848690922
0.274472639
1.102307933





similar to actin





related protein 2/3





complex, subunit 3





(21 kDa); Arp2/3





complex subunit





p21-Arc [Mus





musculus]





[M. musculus]


1388683_at
AI411174

ESTs, Highly
8.66963072
8.511747
−0.43469
0.896338715
0.466659704
1.111224693





similar to





hypothetical





protein MGC14151





[Homo sapiens]





[H. sapiens]


1375245_at
AA800669

ESTs, Highly
10.09883803
9.917245
−0.40076
0.881728933
0.482696461
1.136988127





similar to A36180





61K transforming





protein - human





[H. sapiens]


1383054_at
BE111631

ESTs, Highly
7.47092638
7.691487
0.684678
1.165186416
−0.438439828
0.895791948





similar to I48724





zinc finger protein





PZF - mouse





[M. musculus]


1389957_at
BG378149

ESTs, Highly
9.604565474
9.852639
0.339593
1.187620315
−0.23684341
0.879305448





similar to JW0059





mtprd protein -





mouse





[M. musculus]


1374593_at
AA799421

ESTs, Highly
8.436415302
8.757416
0.610494
1.249196498
−0.371471138
0.855707419





similar to





KPCE_RAT





PROTEIN KINASE





C, EPSILON TYPE





(NPKC-EPSILON)





[R. norvegicus]


1375119_at
BI284798

ESTs, Highly
9.262421398
9.747071
0.469691
1.399245716
−0.269750886
0.795573103





similar to S70642





ubiquitin ligase





Nedd4 - rat





(fragment)





[R. norvegicus]


1375305_at
BI282028

ESTs, Highly
10.64049686
10.86651
0.543331
1.169598117
−0.407433833
0.884962553





similar to





ST1B_MOUSE





Syntaxin 1B





(P35B)





[R. norvegicus]


1390423_at
BE104245

ESTs, Highly
8.796017809
9.069358
0.231788
1.208602479
−0.333710591
0.783587436





similar to T14792





hypothetical





protein





DKFZp586G0322.





1 - human





(fragment)





[H. sapiens]


1398971_at
BI283725

ESTs, Moderately
9.374401563
9.553263
0.485723
1.13199048
−0.397357886
0.8957128





similar to





KIAA0100 gene





product [Homo





sapiens]





[H. sapiens]


1388850_at
BG671521

ESTs, Moderately
9.638454301
9.957086
0.267318
1.247146979
−0.37021886
0.693183088





similar to





HS9B_RAT Heat





shock protein HSP





90-beta (HSP 84)





[R. norvegicus]


1390592_at
BM389412

ESTs, Moderately
7.927245389
8.209323
0.666237
1.215944997
−0.373953853
0.883872321





similar to T14273





zinc finger protein





106 - mouse





[M. musculus]


1390097_at
BI281738

ESTs, Moderately
9.3184521
9.600735
0.359884
1.216117474
−0.289309137
0.840945647





similar to





Y193_HUMAN





Hypothetical





protein KIAA0193





[H. sapiens]


1371590_s_at
BM386159

ESTs, Weakly
8.508270545
8.290722
−0.40327
0.860025673
0.715808502
1.286471079





similar to e-





Tropomodulin





[Rattus norvegicus]





[R. norvegicus]


1390048_at
BF408990

ESTs, Weakly
7.825368912
8.242474
0.608206
1.335245393
−0.241815196
0.891226221





similar to





hypothetical





protein, MNCb-





4760 [Mus





musculus]





[M. musculus]


1375231_a_at
BI281838

ESTs, Weakly
9.410742003
9.628854
0.530944
1.163210616
−0.387267422
0.888752574





similar to inhibitor





of the Dvl and Axin





complex [Rattus





norvegicus]





[R. norvegicus]


1399079_at
AI101659

ESTs, Weakly
9.836360668
10.16998
0.396804
1.260170799
−0.239640185
0.859334251





similar to SC65





synaptonemal





complex protein





[Rattus





norvegicus]





[R. norvegicus]


1388903_at
AI179335

ESTs, Weakly
8.332638336
8.114142
−0.5078
0.859460826
0.35815011
1.110966168





similar to t-





complex testis





expressed 1





[Rattus norvegicus]





[R. norvegicus]


1373063_at
BI277000

ESTs, Weakly
8.697615098
8.502515
−0.43031
0.87351232
0.463990102
1.15709537





similar to ubiquitin-





conjugating





enzyme E2N





(homologous to





yeast UBC13);





bendless protein





[Rattus norvegicus]





[R. norvegicus]


1371337_at
BG378939

ESTs, Weakly
9.813332563
9.642615
−0.3672
0.888400916
0.325054592
1.106521553





similar to S13099





cytochrome-c





oxidase (EC





1.9.3.1) chain Vlla





precursor - rat





[R. norvegicus]


1398846_at
BE107346
56783
eukaryotic initiation
8.361376446
9.008593
0.495006
1.566143804
−0.414497851
0.664667317





factor 5 (elF-5)


1387383_at
NM_031802
83633
G protein-coupled
9.961560372
9.722226
−0.41601
0.847135806
0.740340744
1.292462812





receptor 51


1368401_at
M85035
29627
glutamate
9.7450637
10.12056
0.388163
1.297283064
−0.370340093
0.761334957





receptor,





ionotropic, 2


1388189_at
AW522430
24416
glutamate
7.922810149
8.369635
0.603662
1.363036876
−0.282310414
0.857046994





receptor,





metabotropic 3


1387659_at
AF245172
83585
guanine
8.579875817
8.970425
0.419618
1.310892245
−0.310557432
0.801387052





deaminase


1375705_at
AI103622
24400
Guanine
11.052428
11.43942
0.624776
1.307666247
−0.713058008
0.698548881





nucleotide-binding





protein beta 1


1370053_at
BE116953
65040
guanylate kinase
8.181514198
8.416934
0.402124
1.177248901
−0.304521974
0.873239295





associated protein


1375532_at
AI008792
25587
Inhibitor of DNA
8.470555381
9.278552
0.655617
1.750778223
−0.467889277
0.6448088





binding 2,





dominant negative





helix-loop-helix





protein


1371148_s_at
X52017
24503
internexin, alpha
8.348395102
8.742675
0.442247
1.314286384
−0.351712988
0.790245803


1370865_at
BI277627
25179
isocitrate
9.508280943
9.334029
−0.41328
0.886227042
0.430076542
1.117948633





dehydrogenase 3,





gamma


1387071_a_at
BE107978
29477
microtubule-
10.20988985
11.06574
0.440253
1.809820398
−0.40915131
0.566925482





associated





protein tau


1370831_at
AY081195
29254
monoglyceride
7.997950072
8.519811
0.691492
1.435806237
−0.370997892
0.797665306





lipase


1370016_at
NM_031070
81734
nel-like 2
9.366440831
9.130241
−0.3398
0.84897858
0.629322922
1.313198487





homolog





(chicken)


1369690_at
AI547471
60355
N-ethylmaleimide
9.871860703
9.66198
−0.40131
0.864608826
0.356371725
1.111739806





sensitive factor


1368993_at
NM_020088
56762
neurestin
7.578732705
8.063511
0.624639
1.399370323
−0.459690254
0.755545133


1369404_a_at
NM_021767
60391
neurexin 1
7.850829037
8.133737
0.400729
1.216644537
−0.47177386
0.798122143


1370058_at
NM_031783
83613
neurofilament, light
9.844994746
9.631533
−0.42188
0.862465188
0.366920816
1.10425063





polypeptide


1370517_at
U18772
266777
neuronal pentraxin 1
9.516316885
9.313932
−0.51492
0.869112366
0.522032631
1.16884432


1368255_at
NM_017354
50864
neurotrimin
8.456459057
8.628735
0.372202
1.126834366
−0.377704419
0.883893225


1367851_at
J04488
25526
Prostaglandin D
11.49214354
11.29404
−0.3933
0.871692914
0.290393494
1.108310809





synthase


1398790_at
NM_017039
24672
Protein
9.819878116
9.61136
−0.34567
0.865425423
0.311060317
1.132692891





phosphatase 2





(formerly 2A),





catalytic subunit,





alpha isoform


1398825_at
D01046
79434
RAB11B, member
8.467885101
8.303234
−0.51417
0.892144413
0.494808325
1.119497348





RAS oncogene





family


1370087_at
NM_031718
65158
RAB2, member
8.267117217
8.013845
−0.58443
0.838991442
0.408100105
1.123112511





RAS oncogene





family


1370372_at
AF134409
171099
RASD family,
8.98764962
8.822419
−0.40481
0.891785751
0.417570782
1.12716855





member 2


1369816_at
NM_013018
25531
Ras-related small
9.593343909
9.380739
−0.35073
0.862977618
0.546884115
1.248974009





GTP binding





protein 3A


1369958_at
NM_022542
64373
rhoB gene
8.970515013
8.798449
−0.38451
0.887570983
0.423174482
1.123272591


1375421_a_at
AI600019
192256
rotein carrying the
9.594165696
10.03902
0.3913
1.361178681
−0.323072636
0.74473796





RING-H2





sequence motif


1375621_at
AI575254
261737
sideroflexin 5
7.407218485
7.669141
0.495738
1.199075642
−0.417215583
0.848997619


1370224_at
BE113920
25125
signal transducer
7.635173326
7.857439
0.709037
1.166564469
−0.459754483
0.890983344





and activator of





transcription 3


1388000_at
AF021923
84550
solute carrier
7.301200621
7.512385
0.309079
1.157637879
−0.25114173
0.870463115





family 24





(sodium/potassium/





calcium





exchanger),





member 2


1368440_at
NM_017216
29484
solute carrier
9.398846616
9.93355
0.749691
1.448643968
−0.727328652
0.650057604





family 3, member 1


1389986_at
AI008409
117556
synaptic vesicle
8.015724584
8.590117
0.656167
1.489050598
−0.357264595
0.775227175





glycoprotein 2 b


1369627_at
L10362
117556
synaptic vesicle
8.663172726
8.82176
0.204444
1.116193334
−0.243485248
0.864656842





glycoprotein 2 b


1387662_at
L38247
64440
synaptotagmin 4
8.677669502
9.157806
0.309263
1.394875371
−0.334811005
0.692224654


1369879_a_at
NM_019381
24822
Testis enhanced
8.733602786
8.580479
−0.38722
0.899301003
0.378082787
1.115748654





gene transcript


1368841_at
NM_053369
84382
transcription
8.639946736
8.85393
0.486185
1.159885847
−0.374241086
0.892103037





factor 4


1386999_at
BG380730
56011
tyrosine 3-
8.669827204
8.515457
−0.44737
0.898524607
0.533133344
1.130282208





monooxgenase/





tryptophan 5





monooxgenase





activation protein,





beta polypeptide


1398843_at
AI411103
58857
vesicle-associated
10.04441626
9.889542
−0.34531
0.898210852
0.35733192
1.10078515





membrane protein,





associated protein a


1386909_a_at
AF268467
83529
voltage-dependent
8.025008371
7.83859
−0.4422
0.87878463
0.441711122
1.146114211





anion channel 1

## Claims

1. A method of decreasing anxiety-like behavior in a subject diagnosed with a mood disorder comprising administering to the subject a therapeutically effective amount of FGF2 that is sufficient to decrease anxiety-like behavior in the subject, wherein said FGF2 is administered orally, intravenously, intrathecally, or intraperitoneally.

2. The method of claim 1, wherein the mood disorder is major depression disorder.

3. The method of claim 1 or 2, wherein said therapeutically effective amount is from 1 nanogram to 10 milligrams per kilogram of said subject's body weight.

4. The method of claim 1, wherein said FGF2 is administered with a pharmaceutically acceptable carrier.

5. The method of claim 1, wherein said subject is a human.

6. The method of claim 1, wherein said FGF2 is a recombinant polypeptide.